Protein phosphatase methylesterase

ABSTRACT

Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells. The protein phosphatase 2A (PP2A) catalytic (C) subunit is reversibly methylated at its carboxy-terminus by specific methylesterase. Carboxymethylation affects PP2A activity and varies during the cell cycle. The present disclosure provides the coding sequence of a methylesterase, herein named Protein Phosphatase Methylesterase-1 (PME-1). PME-1 is highly conserved from yeast to human and contains a motif found in lipases, which motif has a catalytic triad-activated serine as the active site nucleophile. Recombinant PME-1 polypeptide produced in bacteria demethylates PP2A C subunit in vitro and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction. PME-1 represents the first mammalian protein phosphatase methylesterase cloned to date.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 09/293,322 filed Apr. 16, 1999 now U.S. Pat. No. 6,232,110 andclaims priority from U.S. Provisional Application Ser. No. 60/082,202,filed Apr. 17, 1998.

ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT

This invention was made, at least in part, with funding from the UnitedStates National Institutes of Health (Grant CA 57327). Accordingly, theUnited States Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

The field of this invention is the area of molecular biology, and inparticular the DNA sequence encoding Protein PhosphataseMethylesterase-1 (PME-1, formerly called p44A), recombinant vectors, andmethods for recombinant production of PME-1 demethylase and its use inidentifying compositions with inhibitory activity.

Protein phosphatase 2A (PP2A) is a highly conserved serine/threoninephosphatase involved in the regulation of a wide variety of enzymes,signal transduction pathways, and cellular events [Cohen, P. (1989)Annu. Rev. Biochem. 58:453-508; Lee, T. H., et al. (1991) Cell64:415-423; Mayer-Jaekel, R. E. et al. (1993) Cell 72:621-633; Sontag,E. S. et al. (1993) Cell 75:887-897; Uemura, T. et al. (1993) Genes Dev.7:429-440]. The minimal structure thought to exist in vivo consists of aheterodimer between a catalytic 36 kDa subunit termed C and a constantregulatory 63 kDa subunit termed A [Kremmer, E. et al. (1997) Mol. CellBiol. 17:1692-1701; Usui, H. et al. (1988) J. Biol. Chem.263:3752-3761]. This heterodimer is often further complexed with one ofseveral additional regulatory subunits termed B, B′, and B″ [Cohen, P.(1989) supra]. In PP2A heterotrimers, the A subunit binds to both thecatalytic C and regulatory B-type subunits [Ruediger, R. et al. (1992)J. Virol. 68:123-129; Ruediger, R. et al. (1994) Mol. Cell Biol.12:4872-4882]. In the case of the B subunit, it has been shown that oneor more of the nine C subunit carboxy terminal amino acids are essentialfor heterotrimer formation [Ogris, E. et al. (1997) Oncogene15:911-917]. In cells stably transformed by the middle tumor antigen(MT) of polyomavirus, MT is found in place of the B subunit in a smallportion (−10%) [Ulug, et al. (1992) J. Virol. 66:1458-1467] of PP2Acomplexes [Pallas, D. C. et al. (1990) Cell 60:167-176]. MT/PP2A complexformation is important for MT-mediated transformation [Grussenmeyer, etal. (1987) J. Virol. 61:3902-3909; Pallas, et al. (1988) J. Virol.62:3934-3940; Glenn, G. M. et al. (1995) J. Virol. 69:3729-3736;Campbell, K. S. et al. (1995) J. Virol. 69:3721-3728]. Unlike for Bsubunit, formation of PP2A heterotrimers containing MT does not requirethe last nine amino acid residues of the C subunit [Ogris, E. et al.(1997) supra]. The small tumor antigens (STs) of various papovavirusesalso form complexes with the A and C subunits of PP2A [Pallas, D. C. etal. (1990) supra].

Consistent with the multiple important roles that PP2A plays in diversepathways and cellular events, PP2A is highly regulated. The regulatorymechanisms include modulation by regulatory subunits or inhibitoryproteins and modulation by post-translational modification of the Csubunit. Subunit composition of the PP2A complex affects both catalyticactivity and substrate specificity [Agostinis, P. et al. (1992) Eur. J.Biochem. 205:241-248; Favre, B. et al. (1994) J. Biol. Chem.269:16311-16317; Scheidtmann, K. H. et al. (1991) Mol. Cell. Biol.11:1996-2003; Sola, M. M. et al. (1991) Biochem. Biophys. Acta1094:211-216]. In the case of B subunit, changes of up to 100 fold havebeen documented using cdc2 phosphorylated substrates [Agostinis, P. etal. (1992) Eur. J. Biochem. 205:241-248; Ferrigno, P. et al. (1993) Mol.Biol. Cell 4:669-677; Mayer-Jaekel, R. E. et al. (1994) Journal of CellScience 107:2609-2618; Ogris, E. et al. (1997) supra; Sola, M. M. et al.(1991) Biochem. Biophys. Acta 1094:211-216]. Two PP2A inhibitor proteinshave been reported: I1PP2A (also called PHAPI) and I2PP2A (also calledPHAPII or SET) [Li, M. et al. (1996) Biochemistry 34:1988-1996; Li, M.et al. (1996) Biochemistry 35: 6998-7002; Li, M. et al. (1995) J. Biol.Chem. 271:11059-11062]. These also appear to be substrate-dependent intheir effects. Perusal of the NCBI GenBank and EST databases via BLASTfollowed by sequence comparisons using DNASTAR MegAlign softwareindicates the existence of three different human PHAPI isoforms encodedby different genes and the presence of multiple alternatively splicedforms of PHAPII. A Xenopus homolog of PHAPII was recently shown tointeract with B-type cyclins in vitro [Kellogg, D. R. et al. (1995) J.Cell Biol. 130:661-673], but the molecular consequences of thisinteraction in the regulation of PP2A are not known.

The post-translational modifications of the C subunit that have beenreported to modulate PP2A activity include phosphorylation andmethylation. Inhibition of PP2A activity in vitro was found upon Csubunit phosphorylation at either tyrosine 307 or at one or moreunidentified threonine residues [Chen, J. et al. (1992) Science257:1261-1264; Guo, H. and Damuni, Z. (1993) Proc. Natl. Acad. Sci. USA90:2500-2504]. A similar modification may occur in vivo in response totransformation or growth stimulation [Chen, J. et al. (1994) J. Biol.Chem. 269:7957-7962]. The first indication that PP2A C subunit wasmethylated involved two observations. A 36 kDa SV40 small tumor antigen(ST)-associated cellular protein is a major acceptor of the methyl groupfrom radiolabeled S-adenosyl methionine added to cell extracts [Rundell,K (1987) J. Virol. 61:1240-1243]. This ST-associated cellular proteinwas reported to be the PP2A C subunit [Pallas, D. C. et al. (1990)supra]. The site of methylation of the PP2A C subunit has beenidentified as leucine 309 [Favre, B. et al. (1994) supra; Lee, J. andStock, J. (1993) J. Biol. Chem. 268:19192-19195; Xie, H. and Clarke, S.(1994) J. Biol. Chem. 269:1981-1984]. One study reported anapproximately two-fold increase in the activity of PP2A uponmethylation, adjusting for the stoichiometry of methylation [Favre, B.et al. (1994) supra]. Only phosphorylase a and the peptide substrate,phosphorylated Kemptide, were used in that study. These substrates oftengive similar results. Thus, it remains to be determined whether greatereffects might be observed with other substrates. Based on differentialantibody recognition of methylated and non-methylated C subunit, PP2Ahas been reported to undergo cell cycle dependent changes in methylation[Turowski, P. et al. (1995) J. Cell Biol. 129:397-410]. It is not knownwhether methylation of PP2A affects the subunit composition of theenzyme. Partially purified fractions of PP2A containing A/C heterodimersor A/B/C heterotrimers have both been shown to be substrates for thePP2A methyltransferase [Xie, H. and Clarke, S. (1994) supra]. There arealso data which indicate that methylated C subunit can associate withSV40 ST [Rundell, K. (1987) supra].

The B subunit functions in cell cycle progression through mitosis and incytokinesis [Healy, A. M. et al. (1991) Mol. Cell Biol. 11:5767-5780;Mayer-Jaekel, R. E. et al. (1993) supra; Uemura, T. et al. (1993) GenesDev. 7:429-440]. In cells stably transformed by the middle tumor antigen(MT) of polyomavirus, MT is found in place of the B subunit in a smallportion (^(˜)10%) [Ulug, E. T. et al. supra] of PP2A complexes [Pallas,D. C. et al. (1990) supra]. MT/PP2A complex formation is known to beimportant for MT-mediated transformation [Campbell, K. S. et al. (1995)supra; Glenn, G. M. et al. (1995) supra; Grussenmeyer, T. et al. (1987)supra; Pallas, D. C. et al. (1988) supra], but the precise functionalconsequences of MT association with PP2A are still being elucidated. Itwas recently shown that there is a requirement for direct B/C subunitinteraction to form stable heterotrimers [Ogris, E. et al. (1997)supra].

The nine carboxy-terminal amino acids of the PP2A C subunit, residues301 to 309, include tyrosine 307, the site of phosphorylation in vitroby v-src, and two potential sites of threonine phosphorylation, residues301 and 304. Seven of these nine residues, including threonine 304 andtyrosine 307, are found in every PP2A C subunit cloned to date.Threonine 301 is somewhat less conserved.

In order to study cellular proteins which interact with PP2A, twocatalytically inactive C subunit mutants were generated and used to formstable complexes. The present invention describes the identification ofone of these proteins, herein named Protein Phosphatase Methylesterase-1(PME-1).

Due to the fact that PP2A is shown to regulate multiple cellularpathways by dephosphorylating several key proteins, there has been along felt need in the art to understand the molecular mechanisms bywhich PP2A activity is modulated. The present invention describescloning of one such modulating enzyme for human PP2A, named hereinPME-1, and also shows how to produce recombinant PME-1 polypeptide,which is then used in in vitro assays to identify inhibitors for PME-1activity.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide nucleotide sequencesencoding protein phosphatase methylesterase-1 (PME-1) and the deducedamino acid sequence therefor. Specifically exemplified coding sequencesare given in Table 2, together with the deduced amino acid sequence forthe human; Tables 6 and 3 for the yeast; Tables 7 and 4 for thenematode. All synonymous coding sequences for the exemplified amino acidsequences are within the scope of the present invention.

It is a further object of the present invention to provide functionallyequivalent coding and protein sequences, including equivalent sequencesfrom other mammals and other organisms, including but not limited toyeast and nematodes, and variant sequences from humans. Functionallyequivalent PME-1 coding sequences are desirably from about 50% to about80% nucleotide sequence homology (identity) to the specificallyidentified PME-1 coding sequence, from about 80% to about 95%, anddesirably from about 95% to about 100% identical in coding sequence tothe specifically exemplified coding sequence. Each integer and eachsubset of each specified range is intended within the context of thepresent invention.

Hybridization conditions of particular stringency provide for theidentification of homologs of the human PME-1 coding sequence from otherspecies and the identification of variant human sequences, where thosehomologs and/or variant sequences have at least (inclusively) 50 to 85%,85 to 100% nucleotide sequence identity, 90 to 100%, or 95 to 100%nucleotide sequence identity.

The PME-1 coding sequence and methods of the present invention includethe homologous coding sequences in organisms other than humans and mice.Methods can be employed to isolate the corresponding coding sequences(for example, from cDNA) from other organisms, including but not limitedto other mammals, avian species, Saccharomyces and Caenorhabditiselegans useful in the methods of this invention using the sequencesdisclosed herein and experimental techniques well known to the art.

It will further be understood by those skilled in the art that othernucleic acid sequences besides those disclosed herein for the PME-1coding sequence will function as coding sequences synonymous with theexemplified coding sequences. Nucleic acid sequences are synonymous ifthe amino acid sequences encoded by those nucleic acid sequences are thesame. The degeneracy of the genetic code is well known to the art. Formany amino acids, there is more than one nucleotide triplet which servesas the codon for a particular amino acid, and one of ordinary skill inthe art understands nucleotide or codon substitutions which do notaffect the amino acid(s) encoded.

Specifically included in this invention are PME-1 sequences from otherorganisms than those exemplified herein, which sequences hybridize tothe PME-1 sequence disclosed under stringent conditions. Stringentconditions refer to conditions understood in the art for a given probelength and nucleotide composition and capable of hybridizing understringent conditions means annealing to a subject nucleotide sequence,or its complementary strand, under standard conditions (i.e., hightemperature and/or low salt content) which tend to disfavor annealing ofunrelated sequences. As specifically exemplified, “conditions of highstringency” means hybridization and wash conditions of 65°-68° C.,0.1×SSC and 0.1% SDS (indicating about 95-100% nucleotide sequenceidentity/similarity). Hybridization assays and conditions are furtherdescribed in Sambrook et al. (1989) Molecular Cloning, Second Edition,Cold Spring Harbor Laboratory, Plainview, N.Y.

As used herein, conditions of moderate (medium) stringency are thosewith hybridization and wash conditions if 50-65° C., 1×SSC and 0.1% SDS(where a positive hybridization result reflects about 80-95% nucleotidesequence identity). Conditions of low stringency are typically thosewith hybridization and wash conditions of 40-50° C., 6×SSC and 0.1% SDS(reflecting about 50-80% nucleotide sequence identity).

As used herein, all or part of a nucleotide sequence refers specificallyto all continuous nucleotides of a nucleotide sequence, or e.g. 1000continuous nucleotides, 500 continuous nucleotides, 100 continuousnucleotides, 25 continuous nucleotides, and 15 continuous nucleotides.

Where PME-1-homologous coding sequences are to be isolated from otherorganisms, one desirably uses nucleotide probes or primers from the mosthighly conserved regions of the PME-1 protein. For example, the skilledartisan desirably uses hybridization probes or PCR primers encoding theactive site region (GHSMGGA, amino acids 154-160, SEQ ID NO:5, in theprotein sequence) and a second highly conserved sequence within theprotein [GQMQGK, amino acids 333-338, SEQ ID NO:5) to derive probe orprimer sequences.

It is well known in the biological arts that certain amino acidsubstitutions may be made in protein sequences without affecting thefunction of the protein. Generally, conservative amino acidsubstitutions or substitutions of similar amino acids are toleratedwithout affecting protein function. Similar amino acids can be thosethat are similar in size and/or charge properties, for example,aspartate and glutamate, and isoleucine and valine, are both pairs ofsimilar amino acids. Similarity between amino acid pairs has beenassessed in the art in a number of ways. For example, Dayhoff et al.(1978) in Atlas of Protein Sequence and Structure, Volume 5, Supplement3, Chapter 22, pp. 345-352, which is incorporated by reference herein,provides frequency tables for amino acid substitutions which can beemployed as a measure of amino acid similarity. Dayhoff et al.'sfrequency tables are based on comparisons of amino acid sequences forproteins having the same function from a variety of evolutionarilydifferent sources.

Also within the scope of the present invention are recombinant hostcells and recombinant vectors carrying the PME-1 coding sequences of thepresent invention. Desirably, those coding sequences are operably linkedto transcriptional and translational control sequences functional in thehost cell into which the vectors are introduced and maintained.

Further provided by the present invention are methods for therecombinant production of a PME-1 protein. After a suitable vector inwhich a PME-1 coding sequence is operably linked to transcriptional andtranslational control sequences is introduced into a recombinant hostcell of choice, the recombinant host cells are cultured under conditionswhere the PME-1 sequences are expressed. The PME-1 can then berecovered, if desired. It is understood that the vector and host cellsare chosen for maintenance of the vector within the host cell.Similarly, the transcriptional and translational control sequences arechosen for function in the host cell of choice. The specificallyexemplified human PME-1 sequence can be modified, for example, usingpolymerase chain reaction (PCR) technology by substituting synonymouscodons according to the known codon usage of the chosen host cell sothat expression of the coding sequence is maximized.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that the catalytically inactive mutants of PP2A can formcomplexes with the regulatory A subunit and MT in vivo. Lysates fromcells containing only control vector (GRE only) or HA-tagged wt (wt-36)or mutant C subunits (H59Q, H118Q) were precipitated with anti-HA tagantibody (12CA5) and analyzed by SDS-PAGE and immunnoblotting. The blotwas probed first with anti-MT antibody, and then sequentially withantibodies recognizing the A, C (via the epitope tag), and B PP2Asubunits. Because a lower level of expression was consistently seen withH118Q, the immunoprecipitate of this mutant was prepared from morecells; to properly control for this, the control immunoprecipitate wasprepared from an equivalent amount of cells expressing only the vector.Under these conditions, a small amount of MT binds non-specifically tothe immunoprecipitate in the GRE only lane.

FIG. 2A illustrates HA tag immunoprecipitates prepared from ³⁵S-labeledcell lines individually expressing HA-tagged wt (36 wt) or mutant Csubunits (H590Q, H118Q) or vector only (GRE only) analyzed by SDS-PAGEand autoradiography. Portions of the gel where C subunit, A subunit, anda novel 44 kDa protein migrate are shown. The C subunits migrate asdoublets in these gels; whether doublets or a single band are seenvaries from gel to gel (compare with FIG. 1). Migration of C subunit asdoublets on SDS-PAGE has been noted previously for both HA-tagged andendogenous PP2A C subunits [Campbell et al. (1995) supra; Ogris et al.(1997) supra; Turowski et al. (1995) supra] and does not appear to bedue to degradation. The panels and lanes shown are from the sameexperiment and gel, but the lanes were not all originally adjacent. Evenon long exposure, the 44 kDa protein seen in the mutant lanes is notseen in the wt or control lanes.

FIG. 2B shows immunoprecipitates identical to those in FIG. 2A analyzedby 2D gel electrophoresis. Only the portion of each gel containing therelevant proteins is shown. The A, B and C subunits and p44B areindicated by labeled brackets and arrowheads, while the correspondingpositions in panels lacking these proteins are indicated with unlabeledbrackets or arrowheads. For reference, actin is indicated in all panelsby a small unlabeled arrow.

FIG. 2C shows silver-stained 2D gels of HA tag immunoprecipitatesprepared from unlabeled cells expressing vector only (GRE only) or the Csubunit mutant, H118Q. Only the portion of each gel containing therelevant proteins is shown. The A and C subunits, PME-1, and anti-HA tagantibody heavy chain (Ab) are indicated by labeled brackets andarrowheads. Unlabeled arrowheads indicate the corresponding positions inthe GRE only control panel. For reference, actin is indicated in bothpanels by a small unlabeled arrow. The approximate position that p44Bwould be located on these gels is indicated by the unlabeled brackets.

FIG. 3A is a schematic of a 2.5 kb human PME-1 cDNA. On the stickdiagram, the positions of the in frame 5′ UTR stop codon (TGA), of thefirst two potential start codons (ATGs), of tandem stop codons (TAGTGA)at the end of the PME-1 ORF, and of the poly A tail (bracket) are shown.The 3′ end of the 3′ UTR, including the position of the poly A tail, wasdeduced by analyzing overlapping PME-1 ESTs; all other regions weredirectly sequenced. The sequence shown extends from the in frame 5′ UTRstop codon (TGA; overlined) to the second possible start ATG (doubleunderlined) (SEQ ID NO: 16). The first possible start ATG (underlinedonce in the sequence shown) was identified as the authentic start sitein vivo by making constructs whose transcription/translation products invitro would start with one or the other of these two ATGs. ³⁵S-labeledin vitro transcription/translation product starting at the first ATG,but not the product starting at the second ATG, comigrated precisely on2D gels with PME-1 from HeLa cell lysates.

FIG. 3B shows that PME-1 mRNA is expressed in different tissues. TotalRNA from the indicated mouse organs was separated by electrophoresis andhybridized with a mouse PME-1 partial cDNA probe from the 3′ UTR ofmouse PME-1. In a separate experiment, the size of the PME-1 transcriptwas calculated to be 2.6±0.2 kB. The lower panel shows the 18S rRNA fromthe same blot visualized with methylene blue.

FIG. 4 demonstrates that PME-1 stably associates with H59Q but notwild-type C subunit. HA tag immunoprecipitates prepared from NIH3T3(NIH) or MT-transformed NIH3T3 (NIHMT) cell lines individuallyexpressing HA-tagged wt (wt C sub) or mutant (H59Q) C subunits wereanalyzed by SDS-PAGE and immunoblotting with HA tag antibody and PME-1anti-peptide antibody. The C subunits migrate as tight doublets in thesegels. The panels and lanes shown are from the same experiment and gel,but the lanes were not all originally adjacent. Even on long exposure,the 44 kDa protein seen in the mutant lanes is not seen in the wt lanes.

FIG. 5 shows that human PME-1 is a PP2A methylesterase.Immunoprecipitated PP2A C subunit was incubated with lysates frombacteria either not expressing PME-1 (control) or expressing PME-1(PME-1), or with purified bacterially-expressed PME-1 (^(˜)5 ng).Okadaic acid (O.A.) or PMSF was added to the reactions to the indicatedfinal concentrations. Reactions containing 1.25% DMSO as a control tomatch the level resulting from addition of okadaic acid or PMSF stocksolutions is noted. After incubation, the immunoprecipitated PP2A Csubunits were analyzed by SDS-PAGE. Proteins were transferred tonitrocellulose and the membrane was probed with 4b7(methylation-sensitive Ab), an anti-C subunit antibody that onlyrecognizes unmethylated C subunits. Subsequently, the same membrane wasprobed with Transduction Laboratories, (Lexington, Ky.) anti-PP2A Csubunit antibody (methylation-insensitive Ab), which is insensitive tothe methylation state of PP2A and therefore reveals the total C subunitin each lane. The C subunits migrated as doublets in this gel, butwhether double or single bands are seen can vary (see comments in legendto FIG. 2A).

FIG. 6A shows that the PP2A inhibitors, okadaic acid, sodium fluoride,and sodium pyrophosphate, reduce the amount of PME-1 complexed with thecatalytically inactive H59Q C subunit. Seven parallel dishes of NIH3T3cells expressing HA-tagged H59Q were lysed in NP40 lysis buffercontaining the indicated inhibitor(s) at the following concentrations:sodium vanadate (1 mM); NaF (50 mM); okadaic acid (500 nM);phenylarsineoxide (PAO; 10 μM); sodium pyrophosphate (Na₄P₂O₇; 20 mM).Anti-HA tag immunoprecipitates were prepared from these lysates andanalyzed by SDS-PAGE and immunoblotting. The blot was probedsequentially with antibodies detecting PME-1 and H59Q C subunit (via itsHA tag). In a separate experiment using phosphorylase a as substrate,sodium fluoride, okadaic acid and sodium pyrophosphate were respectivelyfound to inhibit PP2A 91±10%, 97±4%, and >99%, while phenylarsineoxideand sodium vanadate respectively showed no or 25±18% inhibition.

FIG. 6B shows that loss of the C subunit carboxy-terninus reduces, butdoes not abolish, PME-1 Binding. Non-immune (N) and HA tag (I)immunoprecipitates were prepared from MT-transformed NIH3T3 cellsexpressing vector only (GRE only), HA-tagged H59Q, or HA-taggedH59Q/301Stop double mutant which lacks nine carboxy-terminal aminoacids. Immune complexes were analyzed by SDS-PAGE; proteins weretransferred to nitrocellulose; and immunoblotting was performed withantibodies directed against A subunit, PME-1, and C subunit (anti-HAtag). The C subunits migrate as doublets in this gel, but whether doubleor single bands are seen can vary (see comments in legend to FIG. 2A).The band seen in all lanes in the PME-1 panel is from theimmunoprecipitating antibodies. Chemiluminescent quantitation (using aBiorad Fluor-S Max Multiumager, Hercules, Calif.) was used in sevenseparate experiments with mixtures of clones to quantify the ratio ofPME-1 to C subunit signal in each lane. In six of seven experiments withmixes of clones, the double mutant bound less PME-1 than did H59Q, witha mean reduction of 56±30% and a median value of 39 (range of 8-87%).Thus, PME-1 binding is clearly reduced by loss of the carboxy-terminus.In a seventh experiment, for unknown reasons, the double mutant bound235 % of the H59Q level of PME-1, lowering the overall mean reduction to28% (median=40).

FIG. 6C demonstrates that subunit carboxy-terminal antibodiesimmunoprecipitate reduced amounts of H59Q/PME-1 Complex.Immunoprecipitates were prepared from MT-transformed NIH3T3 cellsexpressing HA-tagged H59Q using control antibody, HA-tag antibody(12CA5), or carboxy-terminal C subunit antibodies (1D6, 4B7, 4E1). Theimmune complexes were analyzed by SDS-PAGE; proteins were transferred tonitrocellulose; and immunoblotting was performed with anti-A subunitantibody (upper panel), anti-PME-1 antibody (middle panel) and anti-Csubunit antibody recognizing both endogenous and HA tagged proteins(1D6; lower panel). The positions of A subunit, the immunoprecipitatingantibody heavy chains (Ab), PME-1, HA-tagged H59Q C subunit, anduntagged, endogenous wt C subunit are indicated. The C subunits migrateas single bands in this gel, but whether double or single bands are seencan vary (see comments in legend to FIG. 2A). HA-tagged H59Q C subunitmigrates more slowly than endogenous wt C subunit because of the HA tag.

DETAILED DESCRIPTION OF THE INVENTION

“Nucleic acids” and “polynucleotides,” as used herein, may be DNA orRNA. One of skill will recognize that the sequences from nematode genesused in the methods of the invention need not be identical and may besubstantially identical (as defined below) to sequences disclosed here.In particular, where a polynucleotide sequence is transcribed andtranslated to produce a functional polypeptide, one of skill in the artrecognizes that because of codon degeneracy, a number of synonymouspolynucleotide sequences will encode the same polypeptide. Similarly,because amino acid residues share properties with other residues,conservative substitutions of amino acids within a polypeptide may leadto distinct polypeptides with similar or identical function.

The term “operably linked” refers to functional linkage, for example,between a promoter and a downstream sequence, wherein the promotersequence initiates transcription of the downstream sequence.

“Percentage of sequence identity” for polynucleotides and polypeptidesis determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e. gaps) as compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Gaps introduced tooptimize alignment are treated as mismatched, whether introduced in thereference sequence or the comparison sequence. Optimal alignment ofsequences for comparison may be conducted by computerized implementationof known algorithms (e.g. GAP, BESTFIT, FASTA, and TFASTA in theWisconsin Genetics Software Package, Genetics Computer Group (GCG), 575Science Dr., Madison, Wis., or BlastN and BlastX available from theNational Center for Biotechnology Information), or by inspection.Sequences are typically compared using either BlastN or BlastX withdefault parameters.

Substantial identity of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 75% sequenceidentity, preferably at least 80%, more preferably at least 90% and mostpreferably at least 95%. Typically, two polypeptides are considered tobe substantially identical if at least 40%, preferably at least 60%,more preferably at least 90%, and most preferably at least 95% areidentical or conservative substitutions. Sequences are preferablycompared to a reference sequence using GAP using default parameters.

Polypeptides which are “substantially similar” share sequences as notedabove except that residue positions which are not identical may differby conservative amino acid changes. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a group of amino acids having aliphatic sidechains of amino acids having aliphatic-hydroxyl side chains is serineand threonine; a group of amino acids having amide-containing sidechains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups include but are not limited to:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, asparagine-glutamine, and aspartate-glutamate.

Another indication that polynucleotide sequences are substantiallyidentical is if two molecules selectively hybridize to each other understringent conditions. Stringent conditions are sequence dependent andwill be different in different circumstances. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength and pH.The Tm is the temperature (under defined ionic strength and pH) at which50% of the target sequence hybridizes to a perfectly matched probe.Typically stringent conditions for a Southern blot protocol involvewashing at 65° C. with 0.2×SSC.

The present inventors have disclosed the full length cDNA encoding humanprotein phosphatase methylesterase-1, termed PME-1 herein.

Two PP2A C subunit mutants with single amino acid changes in theiractive site residues were found to form stable complexes with cellularproteins. Mutation of either of two histidines predicted to be in thePP2A C subunit active site results in a stable complex between themutant C subunit and a protein of 44 kDa. This 44 kDa protein (formerlycalled p44A) is termed PME-1 herein. Immunoaffmity purification of Csubunit/PME-1 complexes generated sufficient protein for microsequencingof HPLC purified PME-1 tryptic peptides. Three of the nine peptidesequences matched a human Expressed Sequence Tag (EST), which thepresent inventors teach consists of the 3′ end of the PME-1 codingregion and the entire 3′ untranslated sequence. The complete codingregion of the human PME-1 cDNA was obtained via an approach involvingnested and semi-nested polymerase chain reaction (PCR), utilizing 3′primers corresponding to PME-1 EST sequence and 5′ primers correspondingto vector sequence flanking inserts in cDNA libraries. The PME-1 proteinwas identified as the PP2A methylesterase by several criteria, includingmolecular size, presence of a motif found in esterases (includinglipases) utilizing serine as the nucleophilic catalytic residue, abilityof okadaic acid (a known inhibitor of both PP2A and the PP2Amethylesterase) to inhibit association of PME-1 with the C subunitmutants and to inhibit PME-1 activity, and finally, activity assaysperformed in vitro with bacterially expressed protein. Complex formationof PME-1 and mutant C subunit involves, at least in part, the C subunitcarboxy terminus. A catalytically inactive C subunit lacking thecarboxy-terminal 9 amino acids showed decreased association with themethylesterase, and an antibody specific for the C subunit C-terminus,whose binding is sensitive to mutation of tyrosine 307, interfered withPME-1 binding. Finally, the two mutants that complex with PME-1 do notbind substantial amounts of B subunit. However, two other catalyticallyinactive mutants that do not bind PME-1 also are deficient in B subunitbinding.

The carboxy terminus of the protein phosphatase 2A (PP2A) catalytic (C)subunit is highly conserved. Seven of the last nine residues (301-309)are completely invariant in all known PP2As. Included in these invariantresidues are the known pp60^(c-src) phosphorylation site, tyrosine 307,and the known site of methylation, leucine 309. Additionally, one ormore of the nine carboxy terminal residues is necessary for formation ofPP2A heterotrimers containing the B regulatory subunit. The importanceof this tyrosine for binding the methylesterase, the same change inwhich did not dissociate B subunit, suggests that this is the reason itis so highly conserved.

In order to create catalytically inactive PP2A C subunit mutants thatretained maximum structural integrity, single residues likely to beinvolved in catalysis were mutated conservatively. To identify residuespotentially involved in catalysis, an alignment of PP2A and variousrelated phosphatases was performed to identify highly conservedresidues. A small number of residues were found that are identical inPP2A, PP1, PPX, PP2B, and PPλ. Of those, two histidines (H) at positions57 and 118 were chosen as having catalytic potential, and wereindividually mutated to glutamine (Q), yielding the mutants H57Q andH118Q. Subsequent to the construction of these mutants, the crystalstructures of PP1 and PP2B [Goldberg, J. et al. (1995) Nature376:745-753; Kissinger, C. R. et al. (1995) Nature 378:641-644] and amutational analysis of PPλ [Zhuo, S. et al. (1994) J. Biol. Chem.269:26234-26238] were reported, the results of which implicated thesetwo histidines in PP2A catalysis. As described herein below, each Csubunit mutant cDNA was constructed with the hemagglutinin (HA) tag atits amino terminus to allow for immunoprecipitation analysis [Ogris, E.et al. (1997) supra]. Individual mutants, wild-type C subunit, or norecombinant C subunit (vector only) were expressed stably in NIH3T3 celllines with and without coexpression of MT. In the MT expressing cells,most PP2A complexes still contain B subunit because MT is produced at alow level relative to PP2A.

After construction of stable lines, the C subunit mutants werecharacterized with respect to two properties: 1) ability to formcomplexes containing the A and B subunits or MT and 2) catalyticactivity. To examine complex formation in vivo, immunoprecipitates ofepitope-tagged wt and mutant C subunits were probed by immunoblottingfor the presence of additional subunits and MT (FIG. 1). Both mutantsbind substantial A subunit. H118Q also binds a small amount of Bsubunit, while H59Q binds almost none of this subunit. Although a smallamount of MT was found in control immunoprecipitates, levels of MT wellabove this were readily detected in the mutant immunoprecipitates,indicating that A/C/MT trilneric complexes had been formed by theseproteins. A portion of the MT coimmunoprecipitated with H59Q is shiftedrelative to the MT associated with wt C subunit; this result isreproducible and will be described in more detail elsewhere. Theseresults indicate that both of these mutants have substantial nativestructure in vivo.

To test for catalytic activity, phosphatase assays were performed onanti-tag immunoprecipitates from the various cell lines. Using bothphosphorylase and histone H1 as substrates, only wt C subunitimmunoprecipitates were found to have increased activity as compared tocontrol immunoprecipitates prepared from a cell line containing only“empty” vector (Table 1). Immunoprecipitates of the two mutants showedno activity over background towards either substrate. This finding isconsistent with previous published results for mutation of thecorresponding residues in related phosphatases.

Catalytically inactive mutants have the potential to form stablecomplexes with physiological substrates. To determine if novel cellularproteins associated with one or both catalytically inactive C subunitmutants, anti-tag immunoprecipitates were prepared from ³⁵S-labeledcells. FIG. 2A shows that, in addition to the presence of the C and Asubunits, a protein of 44 kDa (p44B) is present in theimmunoprecipitates of both catalytically inactive mutants. More p44Bappears to associate with H59Q than with H118Q. This protein is notpresent in immunoprecipitates prepared from either cells expressing wt Csubunit or cells containing only “empty” vector. The p44B proteinmigrates slightly slower than the non-specific actin band which can beseen in all lanes, and actually overlaps the actin bands in this gel. Ontwo-dimensional (2D) gels, however, p44B is completely separated fromactin and forms a streak with a pI near 7.

In order to see if sufficient p44B could be obtained to facilitatemicrosequencing, scaled up immunoprecipitates were analyzed on 2D gelsand silver-stained. FIG. 2C shows silver-stained 2D gels ofimmunoprecipitates from vector only control cells (GRE only) and fromcells expressing H118Q. P44B was not readily visible in these gels (seebrackets); however, another 44 kDa protein was seen that alsospecifically coimmunoprecipitates with H118Q. This protein, nowdesignated PME-1, was present in almost a 1:1 stoichiometry with the Aand C subunits and was formerly called p44a because its pI,approximately 6, was more acidic than that of p44B. A similar PME-1 spotwas found in silver-stained immunoprecipitates of H59Q. Comparison ofthe H118Q panels in FIG. 2C and FIG. 2B fails to reveal an ³⁵S-labeledspot corresponding to PME-1, suggesting that PME-1 probably has a muchlonger half-life than the PP2A C or A subunits or p44B.

To facilitate cloning of the nucleotide sequence encoding PME-1,sufficient PME-1 protein for microsequencing was obtained by purifyingepitope-tagged H59Q complexes on an anti-tag immunoaffinity column asdescribed hereinbelow. Because PME-1 migrated close to actin on standard10% SDS-PAGE, the separation of these two proteins was optimizedempirically, resulting in the use of a lower percent acrylamideelectrophoresed for an extended period of time. Proteins in the gel wereelectrophoretically transferred to PVDF membrane and visualized bystaining with Ponceau S. Both the actin and a clearly separated 44 kDaband migrating just above it were excised for further processing.Microsequencing of a tryptic peptide from the lower band confirmed thatit was indeed actin. Nine microsequences obtained from the 44 kDa bandmatched no known protein in GenBank, indicating that it was a novelprotein. However, a human EST sequence (H12112) was found deposited thatmatched three of the partial sequences obtained from the 44 kDa protein.In addition, homologous sequences were found in Caenorrhabitis eleganscosmids, and a single Saccharomyces cerevisiae homolog was identified.Additional DNA sequencing of this EST revealed coding information fortwo more PME-1 microsequences, and it was determined that H12112 encodedmost of the carboxy-terminal half of the PME-1 protein (162 aminoacids). Because the EST came from an oligo dT-primed cDNA library, itlikely contains the entire 3′ untranslated region (UTR).

To obtain the missing 5′ portion of the coding region, nested andseminested PCR was performed as described in the Examples hereinbelow.5′ primers corresponded to vector sequence that flanked cDNA inserts inthe library being used as template, and 3′ primers corresponded to knownsequence (EST or newly derived 5′ sequence). In this manner, theremainder of the coding region and a portion of the 5′ UTR wereobtained. Because of the possibility of PCR errors during the multiplereamplification reactions that were necessary to obtain the completecDNA, the sequences of selected portions of the cDNA sequence wereverified. For this purpose, RT-PCR was performed with 5′ and 3′ UTRprimer sequences to generate directly from HeLa cell MRNA a productcontaining the entire coding region and much of the 5′ UTR. The finalcDNA sequence is shown in Table 2.

A schematic of a PME-1 cDNA that includes the end of the 3′ UTR deducedfrom overlapping ESTs is shown in FIG. 3A. The complete cDNA isapproximately 2500 nucleotides in length, including an 1164 nucleotideregion (including tandem stop codons) encoding a protein of 386 aminoacids and a predicted pI of 5.8. All nine tryptic peptide microsequencesobtained from the purified 44 kDa band are found encoded in the clonedcoding sequence throughout its length (underlined in Table 2),confirming that this is the cDNA for the purified 44 kDa associatedprotein. This result is also consistent with the reading frame beingcorrect throughout. There is an in frame stop codon a short distance 5′of the first ATG that was verified in the RT-PCR product, so (withoutwishing to be bound by theory), we believe there is no missing 5′ codingsequence. In addition, the entire coding sequence, including thepositions of the stop codon(s), has been verified several times. Over 98% of the microsequenced murine residues (107 of 109) were identical tothe human sequence. The double underlined serine at position 42corresponds to a threonine in murine PME-1. When a probe specific formouse PME-1 was used to detect transcripts from different mouse organs,a single transcript of _(˜)2.6 Kb was detected in all tissues (FIG. 3B).To date, multiple ESTs have been deposited which encode portions ofPME-1. These sequences separately cover the entire 3′ and 5′ UTRs, butnot the entire coding region, and there is no association between theEST sequences and the function of the encoded protein. Information fromthe NCBI Cancer Genome Anatomy Project (CGAP) indicates that PME-1 ESTshave been mapped to human chromosome 11, interval D1S916-D11S911 (80-84cM). It is not known at this time whether PME-1 is mutated in any of thediseases with defects mapped to this general region of chromosome 11.

The 386 amino acid PME-1 protein product encoded by the human PME-1 cDNAORF is shown in Table 2. It has a pI of 5.8, consistent with itsmigration on 2D gels like the one shown in FIG. 2C. All nine mouse PME-1tryptic peptide sequences (underlined in Table 2) were accounted for inthe human sequence with differences present only at a few positions,indicating that PME-1 is well conserved between these two species. Usingthe NCBI BLAST program, highly homologous sequences probablycorresponding to PME-1 homologs were found for zebrafish, for C.elegans, and for S. cerevisiae. The hypothetical 88.4 kDa C. elegansprotein in chromosome 3, B0464.7, contains some of the C. eleganssequence homologous to PME-1, but lacks other highly homologoussequences, suggesting that it may represent an inaccurate prediction ofexon combinations. A more likely combination of exons that includes allB0464 cosmid exons homologous to PME-1 generates a protein of 365 aminoacids and approximately 40 kDa (Table 2). S. cerevisiae PME-1 (Table 9,Table 3) appears to be a single hypothetical 44.9 kDa protein (PIRaccession number S46814; SwissProt accession number P38796) of unknownfunction encoded by an ORF on chromosome 8R (YHN5; GenBank accessionnumber U10556). Recently YHN5 was proposed to be a mitochondrialribosome subunit protein and named YmS2, based on a single partiallyhomologous nonapeptide sequence [Kitakawa, M. et al. (1997) Eur. J.Biochem. 245:449-456]. Human PME-1 has approximately 40% and 26%respective amino acid identity with the C. elegans and yeast sequences(Table 9). A highly charged stretch of amino acids is present in humanPME-1 but absent in PMEs from C. elegans and S. cerevisiae. This stretchof amino acids does not represent a cloning artifact, because 2D gelcomigration experiments showed that ³⁵S-labeled PME-1 in vitrotranscription/translation product comigrated precisely on 2D gels withPME-1 from HeLa cell lysates.

In order to facilitate further experiments characterizing PME-1, ananti-PME-1 peptide antibody was raised to a sixteen amino acid peptidesequence encoded by the PME-1 cDNA. This peptide antibody detected a 44kDa protein present in H59Q immunoprecipitates, but absent fromimmunoprecipitates of wild-type C subunit (FIG. 4). Thus, PME-1, likep44B, associates stably with the catalytically inactive mutant Csubunits, but not with wt C subunit. Because B subunit, but not MT,requires the C subunit carboxy-terminus for association with the PP2AA/C heterodimer, we wanted to determine if MT expression might increasethe amount of PME-1 bound to H59Q. Similar levels of PME-1 werecoimmunoprecipitated from untransformed NIH3T3 cells and polyomavirusMT-transformed NIH3T3 cells (FIG. 4), indicating that MT expression doesnot greatly affect the level of H59Q/PME-1 complex formation in thecell.

When the human, C. elegans, and S. cerevisiae PME-1 protein sequenceswere analyzed for motifs found in the Prosite database using DNASTARLasergene software, a consensus sequence([LIV]-x-[LIVFY]-[LIVST]-G-[HYWV]-S-x-G-[GSTAC]) (SEQ ID NO: 15) forlipases utilizing an active site serine was found to be conserved. Theinvariant serine in this motif, corresponding to serine 156 in humanPME-1, is the active site serine in these enzymes. In addition,scattered similarities can be seen between other regions of the PME-1sequence and some of the lipases that have this motif. Therefore, PME-1is probably a lipase whose active site serine is serine 156.

The various lipases that share this motif are found in both prokaryotesand eukaryotes and include, among others, two D. melanogastercarboxylesterases. In addition, CheB, a bacterial glutamatemethylesterase, has a similar, but not identical, sequence surroundingits active site serine [Krueger, J. K. et al. (1992) Biochim. Biophys.Acta. 1119:332-326] (Table 8). CheB [West, A. H. et al. (1995) J. Mol.Biol. 250:276-290] and other lipases utilizing an active site serine[e.g. Winkler, F. K. et al. (1990) Nature 343:771-774; Brady, L., et al.(1990) Nature 343:767-770] have a catalytic triad in their primarysequence in the order Ser-Asp(or Glu)-His. Of the conserved histidinesin human PME-1, His 349 is a likely candidate for a putative catalytictriad histidine (Table 9). Identification of a putative PME-1 catalytictriad acidic residue by sequence comparison is more problematic becausethere are multiple acidic residues conserved between species. However,of these, two aspartates in human PME-1, Asp 181 and Asp 324, showconservation in position with putative catalytic triad aspartates inother lipases, and therefore may be more likely possibilities.

A PP2A C subunit carboxyl methylesterase of 46 kDa has recently beenpurified [Lee, J. et al., (1996) Proc. Natl. Acad. Sci., USA93:6043-6047] but no sequence information was reported. To test thepossibility that PME-1 might be a PP2A methylesterase, PME-1 wasexpressed in bacteria and bacterial lysates were tested formethylesterase activity towards PP2A C subunit as described in theExamples herein. The results shown in FIG. 5 demonstrate that lysates ofbacteria expressing PME-1 contain a PP2A methylesterase activity notfound in bacterial lysates lacking PME-1. Similar results were obtainedwith purified recombinant PME-1 (FIG. 5). These results indicate thatPME-1 is indeed a PP2A methylesterase. Because its specificity towardsother methylated phosphatases (such as PPX) has not been characterized,it was generically named Protein Phosphatase Methylesterase-1 (PME-1).

The 46 kDa PP2A methylesterase reported by Lee and coworkers wasinhibited by okadaic acid, a potent PP2A inhibitor, but not by PMSF, acovalent inhibitor of certain serine esterases. To determine if PME-1displays similar sensitivities to these inhibitors, the abovedemethylation assay was also conducted in the presence of okadaic acidand PMSF (FIG. 5). The methylesterase activity of bacterially expressedPME-1 was inhibited by 0.1 or 1 μM okadaic acid but not by 1 or 5 mMPMSF, similar to the methylesterase purified by Lee et al. (1996) supra.

Because single amino acid changes in the C subunit active site werecapable of inducing stable complex formation of C subunit with PME-1, itwas of interest to determine if PP2A inhibitors could antagonize theH59Q/PME-1 complex. To assay for this possibility, NIH3T3 cellsexpressing epitope-tagged H59Q C subunit were lysed in the presence ofvarious phosphatase inhibitors and H59Q was immunoprecipitated via itsepitope tag. The amount of endogenous, untagged PME-1coimmunoprecipitating in each case was assayed by blotting withanti-PME-1 antibody (FIG. 6A). Inhibitors to which PP2A is highlysensitive (okadaic acid, sodium fluoride, and sodium pyrophosphate), butnot those to which PP2A is less sensitive or insensitive (vanadate andphenylarsineoxide, respectively), decreased the amount of PME-1 bound toH59Q.

A PP2A methylesterase might be expected to make important contacts withcarboxy-terminal residues. However, Lee and coworkers found that PP2Acarboxy-terminal peptides functioned neither as inhibitors nor assubstrates for their 46 kDa PP2A methylesterase, suggesting that, at aminimum, contacts with other parts of the C subunit are essential. Toinvestigate the importance of the H59Q C subunit carboxy-terminus forstable interaction with PME-1, a double mutant, H59Q/301Stop, wascreated. This mutant combines the H59Q mutation, which induces stablebinding of PME-1, with a deletion of the nine C subunit carboxy-terminalacids, 301-309. FIG. 6B shows the results of an immunoprecipitationassay measuring the relative abilities of H59Q and H59Q/301Stop to bindA subunit and PME-1. Deletion of residues 301-309 from wt C subunit haspreviously been found to decrease the amount of A subunit bound [Ogris,E. et al. (1997) supra]. FIG. 6B shows that deletion of these residuesfrom H59Q also reduces the binding of the PP2A A subunit to H59Q. Inaddition, although similar amounts of H59Q and H59Q/301Stop wereimmunoprecipitated in this experiment, the double mutant bound lessPME-1 than did H59Q, indicating that one or more of the deletedcarboxy-terminal residues is important for H59Q/PME-1 complex formation.PME-1 binding was not completely abolished, however, demonstrating thatinteractions also exist between PME-1 and other residues in the Csubunit.

To address the same question via a different approach, we assayed viaimmunoprecipitation whether antibodies directed against the C subunitcarboxy-terminus would compete with PME-1 for binding to H59Q. If anantibody competes with PME-1 for binding to residues on H59Q that areimportant for PME-1 association, that antibody would be expected tocoimmunoprecipitate reduced amounts of PME-1 with H59Q when compared toan antibody that does not compete with PME-1. The carboxy-terminal Csubunit monoclonal antibodies used for this experiment, 1D6, 4B7, and4E1, were recently generated against a 15-residue unmethylatedcarboxy-terminal peptide. These antibodies are unable to efficientlyrecognize a C subunit mutant lacking the carboxy-terminal leucine,indicating that they bind, at least in part, at the verycarboxy-terminus. A positive control monoclonal antibody, 12CA5,immunoprecipitates H59Q via its amino-terminal epitope tag and shouldnot interfere with interactions at the C subunit carboxy-terminus[Ogris, E. et al. (1997) supra]. Comparison of the relative ratios ofthe PME-1 and H59Q bands in FIG. 6C reveals that, relative to 12CA5, 1D6and 4B7 inimunoprecipitate less PME-1 for the same amount of H59Q Csubunit (the band of endogenous, wt C subunit immunoprecipitated by thecarboxy-terminal antibodies can be ignored as wt C subunit does notassociate stably with PME-1). Furthermore, although 4E1immunoprecipitated a substantial amount of H59Q C subunit (withinapproximately two-fold of 12CA5), no PME-1 could be detected even onlong exposures. These results thus further substantiate the conclusionsmade from FIG. 6B. In addition, the fact that 1D6 and 4B7coimmunoprecipitate similar amounts of PME-1, but dramatically differentamounts of A subunit indicates that PME-1 binding does not appear to bedependent on A subunit binding.

The successful identification of the first of a number of cellularproteins that stably associate with catalytically inactive PP2A Csubunit mutants, but not with wt C subunit, is reported herein. Twoproteins of 44 kDa that differ in their isoelectric points, PME-1 andp44B, uniquely associated with two different catalytically inactive Csubunit mutants substituted individually at two different active sitehistidine residues. PME-1 was affinity purified and a cDNA encoding itwas cloned. This protein was identified as a PP2A methylesterase byseveral criteria including 1) molecular size; 2) the presence of a motiffound in lipases that use serine as their nucleophilic catalyticresidue; 3) activity assays performed in vitro with bacteriallyexpressed protein; and 4) the ability of okadaic acid, a known inhibitorof both PP2A and the PP2A methylesterase, to inhibit its activity anddecrease its association with the catalytically inactive C subunitmutant, H59Q.

Based on its molecular size, sensitivity to okadaic acid, and the lackof effect of PMSF on PME-1 activity, PME-1 is likely to be equivalent tothe 46 kDa PP2A methylesterase whose purification and initialcharacterization was recently reported by Lee and colleagues [Lee, J. etal. (1996) supra]. Its insensitivity to PMSF indicates that it is notthe PMSF-sensitive serine esterase/protease activity reported by Xie andClarke [Xie, H. et al. (1994) Biochem. Biophys. Res. Commun.203:1710-1715], which also could remove PP2A carboxymethyl groups. Leeand coworkers (1993 supra) reported that their purified PP2Amethylesterase eluted as two different peaks from an anion exchangecolumn, consistent with either differential modification or theexistence of two closely related isoforms of the enzyme. The amounts ofthese two species were within several fold of each other. Two pieces ofevidence from our studies support the idea that those two forms probablyrepresent differentially modified forms of the enzyme. First, probing ofthe GenBank EST database with the PME-1 cDNA sequence provides noevidence for a closely related PME-1 isoform, even though numerous ESTsare found which correspond precisely to the PME-1 cDNA sequence. Second,Northern blot analysis yielded a single band in multiple organs. Inaddition, we have found via immunoblotting that mammalian PME-1 in celllysates migrates on two-dimensional gels as two spots differing in theirisoelectric point in a manner consistent with a single chargedifference.

The molecular basis of the cell cycle-dependent regulation of PP2A Csubunit methylation is unknown. The poor metabolic labeling of PME-1 inan asynchronous population of cells relative to a number of otherproteins suggests that this protein is quite stable. This result arguesagainst the possibility that cell cycle PP2A methylation is regulated bymodulating the amount of the PP2A methylesterase. Whether PME-1 activityis regulated is unknown. In the case of the bacterial chemotacticresponse, the CheB methylesterase is regulated by phosphorylation[Wylie, D. et al. (1998) Biochem. Biophys. Res. Commun. 151:891-896;Hess, J. F. et al. (1998) Cell 53:79-87] while the methyltransferase isthought to be constitutively active. Lee and coworkers (1996 supra)found no difference in the activity of their two purified forms of PP2Amethylesterase, suggesting that the differential modification likelyresponsible for generating these two forms might not be involved inregulation of activity of this enzyme. It is possible, however, thateffects might be seen under other conditions, or that an additionalprotein(s) may be necessary for an effect to be manifested. In addition,it is possible that more than one modification occurs.

Without wishing to be bound by any particular theory, it is believedthat PP2A methyltransferase and methylesterase enzymes achieve theirspecificity, in part, by interacting with or near the active site of thePP2A C subunit. It was reported previously that neither the PP2Amethyltransferase nor the PP2A methylesterase can recognize shortpeptide substrates corresponding to the C subunit carboxy-terminus.Thus, functional recognition by both these enzymes requires additional Csubunit structure. Additionally, as demonstrated in this study,perturbation of the C subunit active site by either of two differentmutations can stabilize the interaction with the PME-1 methylesterase.Furthermore, PP2A inhibitors have a destabilizing effect on thePME-1/H59Q interaction. Finally, the methyltransferase is inhibited bythe PP2A inhibitors, okadaic acid and microcystin-LR, and themethylesterase is inhibited by okadaic acid (testing for inhibition ofthe methylesterase by microcystin has not been reported). Although ithas been proposed that this inhibition is due to the interaction ofthese inhibitors with carboxy-terminal C subunit residues, the PP2Ainhibitors, sodium fluoride or sodium pyrophosphate, partially or fullydisrupt PME-1/H59Q complexes. The latter effect is more consistent witha role in binding the PME-1 methylesterase for active site residuesand/or metals, or nearby residues sensitive to effects on the activesite. Four separate catalytically inactive PP2A active site pointmutants including the two described in this study, are methylated atless than 3% of the wild-type level in vivo and in vitro. Although webelieve there is interaction with residues and/or metals in or near theactive site, but another equally viable possibility is that mutation ofactive site residues and/or binding of inhibitors has more distanteffects on the C subunit conformation critical for stable complexformation with PME-1.

Contact between the C subunit and PME-1 could be with PME-1 residuesand/or with a phosphorylation site on PME-1. Because H59Q and H118Q arevirtually unmethylated, PME-1 apparently can remain bound to thesemutants in the absence of a methylated carboxy-terminus. At least withH59Q, PME-1's contacts other than on the C subunit carboxy-terminus arestrong enough to result in substantial complex formation in the absenceof the nine carboxy-terminal C subunit residues. This conclusion isfurther supported by the finding that two C subunit carboxy-terminalpeptide antibodies, known to require Leu 309 for efficient binding,immunoprecipitate H59Q/PME-1 complexes. However, the amount of PME-1coimmunoprecipitated by these antibodies was less than thatcoimmunoprecipitated by an antibody recognizing an amino-terminalepitope tag on the C subunit. The latter result and the fact that athird carboxy-terminal C subunit antibody could not imrnunoprecipitateH59Q/PME-1 complexes at all suggest that PME-1 is proximal to the Csubunit carboxy-terminus in the H59Q/PME-1 complex. Moreover, thereduced amounts of PME-1 in complex with the H59Q/301Stop double mutantindicate that carboxy-terminal residues play a role in binding of H59Qto PME-1. The contribution of these residues to the interaction of wildtype C subunit with PME-1 might be even more important in the absence ofthe complex-stabilizing H59Q mutation.

The decreased B subunit binding observed with these mutants might be dueindirectly to lack of methylation at the carboxy-terminus of thesemutants. The fact that H59Q and H118Q bind the structural PP2A A subunitand polyomavirus MT suggests that they are not grossly altered in theirstructure. Two other catalytically inactive point mutants that bind Asubunit and polyomavirus MT, but are highly deficient in methylation arealso deficient in B subunit binding. Given that the B subunit requiresthe C subunit carboxy-terminus for stable complex formation with the A/Cheterodimer, the B subunit might require a methylated carboxy-terminusfor efficient binding to C subunit. An alternate, but not mutuallyexclusive, possibility is that the carboxy-terminus and the active siteare proximal in the three dimensional structure of the C subunit. Thismodel would provide an explanation for how events occurring at thecarboxy-terminus (B subunit binding, methylation, phosphorylation, etc.)can affect the active site (activity, specificity), and vice versa. Inaddition, at least for H59Q and H118Q, PME-1 and B subunit binding mightbe mutually exclusive.

These catalytically inactive C subunit mutants should be useful foridentifying other proteins involved in PP2A signaling. H59Q and H118Qbind multiple proteins not bound stably by wt C subunit. These include,in addition to PME-1, p44B and other proteins not marked, but visible,in FIG. 2B. Interestingly, initial experiments suggest that p44B bindingto H59Q is even more sensitive to phosphatase inhibitors than is PME-1binding. These proteins could be PP2A substrates or other proteins whosebinding is sensitive to the state of the C subunit active site. One ofthese proteins is the same molecular size as the PP2A methyltransferasereported by Lee and colleagues (Lee et al. (1993) supra]. Catalyticallyinactive mutants of dual specificity and tyrosine phosphatases[Gelerloos, J. A., et al. (1996) Oncogene 13:2367-2368; Bliska, J. B. etal. (1992) J. Exp. Med. 176:1625-1630] have been previously usedsuccessfully to identify novel substrates, but unlike PP2A, theircatalytic mechanisms involve the formation of covalent intermediateswith substrates.

PME-1 and p44B differ in several characteristics, suggesting that thesetwo proteins are not simply modified forms of one another. They areseparated from each other on two-dimensional gels by approximately onepH unit, which is unlikely to be accounted for by modification; PME-1forms sharp spots on these gels while p44B migrates as a smear. Inaddition, in vitro translation of PME-1 yields no product migrating atthe position of p44B and we have been unable to detect p44B withantibodies raised against PME-1 sequences.

Finally, because of the high conservation of PP2A with otherphosphatases such as PP1, PPX, PPV, etc., it will be of interest to seeif similar or different cellular proteins bind stably to thesephosphatases when the residues corresponding to PP2A H59 and H118 aremutated to glutamine. One question of special interest is whether thecorresponding catalytically inactive mutants of PPX, which has the samelast four carboxy-terminal amino acids as PP2A and is also methylated atits carboxy-terminal leucine, will also trap PME-1.

The present invention provides the coding sequences for the mammalianPME1 protein, as specifically exemplified by the human coding sequence.This allows the construction of recombinant DNA molecules andrecombinant host cells produced in the laboratory, which molecules andhost cells are used for the recombinant expression of the PME1 proteinand enables assay methods for determining inhibitors of themethylesterase activity of the PME-1 protein, and thus, compounds whichslow the growth of cells, especially neoplastic and/or transformedcells.

Without wishing to be bound by theory, the present inventors proposethat the protein of the present invention is a Protein PhosphataseMethylesterase-1 (PME-1) which removes methyl groups from the PP2Agrowth-regulating protein phosphatase, and that the methylation statusof the catalytic subunit affects activity and thus plays a role ingrowth regulation and normal progression of the cell cycle. See, e.g.,Lee et al. (1996) supra, for a description of the methylesterase andmethods for assay.

Monoclonal or polyclonal antibodies, preferably monoclonal, specificallyreacting with the methylesterase of the present invention encoded by aparticular coding sequence may be made by methods known in the art. See,e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratories; Goding (1986) Monoclonal Antibodies:Principles and Practice, 2d ed., Academic Press, New York.

Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described inSambrook et al. (1989) supra; Maniatis et al. (1982) Molecular Cloning,Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth.Enzymol. 218: Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al. (eds.)(1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth.Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose(1981) Principles of Gene Manipulation, University of California Press,Berkeley; Schleif and Wensink (1982) Practical Methods in MolecularBiology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press,Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization,IRL Press, Oxford, UK; and Setlow and Hollaender (1979) GeneticEngineering: Principles and Methods, Vols. 1-4, Plenum Press, New York.Abbreviations and nomenclature, where employed, are deemed standard inthe field and commonly used in professional journals such as those citedherein.

All references cited in the present application are incorporated byreference herein to the extent that they are not inconsistent with thepresent Specification.

The following examples are provided for illustrative purposes, and arenot intended to limit the scope of the invention as claimed herein. Anyvariations in the exemplified sequences and methods which occur to theskilled artisan are intended to fall within the scope of the presentinvention.

EXAMPLES Example 1 Plasmids and Mutagenesis

Site-directed mutagenesis was performed on a HA-tagged wt C subunit cDNAcloned in the pcDNA I Amp vector [Ogris et al. (1997) supra] using theMuta-Gene Phagemid In Vitro Mutagenesis Kit according to themanufacturer's instructions (Bio-Rad Laboratories, Hercules, Calif.).The entire cDNA of both H59Q and H118Q was sequenced to confirmsuccessful mutagenesis and to ensure that no additional mutationoccurred. Mutant C subunit cDNAs including the HA tag coding sequencewere cloned into the dexamethasone-inducible vector, pGRE 5-2 [Mader,S., and White, J. H. (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607].The construction of a pGRE5-2 vector expressing HA-tagged wt PP2A Csubunit has been previously described [Ogris et al. (1997) supra]. Aninducible vector was chosen to try to minimize the potential deleteriouseffects of wild-type and mutant C subunits (if any) while lines werebeing carried in culture, and to provide for an uninduced control inanalyses of their effects.

Example 2 Cells and Cell Culture

NIH 3T3 lines expressing wt polyomavirus MT and a geneticin resistancegene [Cherington et al. (1986) Proc. Natl. Acad. Sci. USA 83:4307-4311]were transfected by the calcium phosphate precipitation method [Sambrooket al. (1989) supra], and individual clones and mixtures of clonesexpressing wt C subunit (36 wt), H59Q, H118Q, or empty vector (GREonly)were selected and maintained as described previously [Ogris et al.(1997) supra]. H118Q expressed at a level well below that of endogenouswt C subunit, while H59Q expressed at a level equal to or greater thanthe wt level. Although the inducible vector, pGRE5-2, was used toexpress these proteins, their levels were substantial in the absence ofdexamethasone; for this reason, GREonly cells were used as a negativecontrol in this study rather than uninduced wt or mutant C subunitexpressing cells. However, dexamethasone treatment was always used toobtain maximal expression of the C subunits.

Example 3 Radiolabeling of Cells

For metabolic labeling of cells with methionine, subconfluent dishes ofcells were labeled for 5 h with [³⁵S] methionine (300 uCi/ml) in DMEMminus methionine supplemented with 0.5% dialyzed fetal bovine serum.

Example 4 Preparation of Cell Lysates and Immunopreciptation

The details of treating the cells with dexamethasone, preparation ofcell lysates, and immunoprecipation of C subunits have been describedpreviously [Ogris et al. (1997) supra]. For experiments quantitatingPME-1 binding to different mutants (FIG. 6B), immunoprecipates werewashed twice with NP40 lysis buffer, twice with PBS, and once withddH20. Washed immune complexes were used for phosphatase assays oranalyzed by one or two-dimensional gel electrophoresis.

Example 5 One- and Two-dimension I Gel Electrophoresis and Fluorography

SDS-polyacrylamide gel (10% acrylamide) was performed according toLaemmli [Laemmli, U.K. (1970) Nature 227:680-685]. Gels were silverstained by the procedure of Wray et al. [Wray, W. et al. (1981)Biochemistry 118:197-203] except that after electrophoresis the gelswere sequentially incubated 10 min in distilled water (200 ml), 10 minin 95% ethanol (200 ml), 1 h. in 50% methanol (100 ml), and 30 min indistilled water (100 ml) prior to staining.

Example 6 Immunoblotting

Immunoblotting [Towbin, H. et al. (1979) Proc. Natl. Acad. Sci. USA76:4350-4354] was performed with mouse monoclonal anti-tag antibody(16B12; 1:5000 dilution of ascites; BAbCO, Richmond, Calif.); rabbitanti-B subunit antibody (#16; 1:5000); affmity-purified rabbit (R39;1:5000) or mouse monoclonal (4G7; 1 μg/ml) anti-A subunit antibodies;mouse monoclonal anti-C subunit antibody (1D6; 0.25 μg/ml); or rabbitant-PME-1 antibodies (AR2 or E37; see below). Immunoblots were developedwith enhanced chemiluminescences (Amersham, Arlington Heights, Ill.).

Example 7 Phosphatase Assay

Phosphatase activity present in anti-HA tag immunoprecipitates from thedifferent cell lines was assayed using phosphorylase a and Histone H1.[γ-³²P]-labeled phosphorylase a substrate was prepared fromphosphorylase b according to the manufacturer's (GibcoBRL, Gaithersburg,Md.) instructions. Histone H1 was phosphorylated by mitotic p34^(cdc2)purified from Nocodazole arrested HeLa cells as described [Mayer-Jaekelet al. (1994) supra]. Lysates used for immunoprecipitation wereequilibrated according to epitope-tagged C subunit expression levels.Assays were performed at a linear range and with subsaturating amountsof each substrate.

Example 8 Purification and Microsequencing of PME-1

To obtain PME-1 protein for microsequencing, H59Q C subunit complexescontaining PME-1 were immunoaffmity purified. In total, 135 confluent 15cm dishes of MT-transformed NIH3T3 cells expressing HA-tagged H59Q wereneeded to obtain enough PME-1 for microsequencing. Forty-five separateinmunoaffinity purifications were performed on 3 dishes of lysate at atime, reusing the same immunoaffinity matrix at least 15 times. Toprepare the immunoaffinity matrix, anti-HA tag antibody (12CA5; obtainedfrom BAbCO) was chemically crosslinked to protein A-Sepharose beads(Pharmacia, Piscataway, N.J.) by published methods [Harlow, E., andLane, D. (1988) supra]. After washing 3 dishes of cells twice with PBSand once with IP wash (10% (vol/vol) glycerol; 135 mM NaCl; 20 mM Tris,pH 8.0), the cells were scraped and lysed at 4° C. with rocking for 10min in 1.0 ml of NP40 lysis buffer (IP wash containing 1% Nonidet P-40,1 mM phenylmethylsulfonyl fluoride, and 0.03 units/ml aprotinin).Lysates were cleared at 13,000×g, and then incubated for 1 h at 4° C.while rocking with 500 μl of the crosslinked antibody/bead complexes.Complexes were washed once with NP40 lysis buffer, three times withTris-buffered saline, and then twice with ddH₂O. Bound H59Q complexescontaining PME-1 were eluted by three sequential incubations with 300 μlof 20 mM triethylamine. Eluates were quickly frozen on dry ice andstored frozen until all batches of affinity purification had beencompleted. The antibody/bead complexes were then washed twice with 20 mMtriethylamine and twice with IPlyse prior to reuse. After H59Q complexeshad been purified from all 135 dishes of cells, eluates containing PME-1were concentrated to dryness by vacuum centrifugation, and the residueswere suspended in PBS and gel buffer and analyzed on three separateSDS-polyacrylamide gels [Laemnmli, U.K. (1970) Nature 227:680-685].One-dimensional gels were chosen to avoid losses associated with 2D gelanalysis. Because PME-1 migrates closely to actin, the separation ofthese two proteins was maximized by the use of an 8% SDS-polyacrylamidegel run for an extended period of time.

Example 9 Trypsin digestion, HPLC separation and microsequencing

After separation of PME-1 complexes by SDS-PAGE, the proteins wereelectrotransferred to polyvinylidiene difluoride (PVDF) membrane andstained with Ponceau S. Individual protein bands were excised andsubmitted to in situ digestion with trypsin [Fernandez et al. (1994)Anal. Biochem. 218:112-117; Lane et al. (1991) J. Protein Chem.10:151-160]. The resulting peptide mixture was separated by microborehigh performance liquid chromatography using a Zorbax C18 2.1 mm by 150mm reverse phase column on a Hewlett-Packard 1090 HPLC/1040 diode arraydetector. Optimum fractions from the chromatogram were chosen based ondifferential UV absorbance at 205 nm, 277 nm and 292 nm, peak symmetryand resolution. Peaks were further screened for length and homogeneityby matrix-assisted laser desorption time-of-flight mass spectrometry(MALDI-MS) on a Finnigan Lasermat 2000 (Hemel, England); and selectedfractions were submitted to automated Edman degradation on an AppliedBiosystems 494A, 477A (Foster City, Calif.) or Hewlett Packard G1005A(Palo Alto, Calif.). Details of general strategies for the selection ofpeptide fractions and their microsequencing have been previouslydescribed [Lane et al. (1991) supra].

Example 10 cDNA Cloning via PCR and RT-PCR

To obtain the missing 5′ portion of the PME-1 coding region, nested andseminested PCR were performed using human B cell, human hippocampus, andhuman kidney cDNA plasmid libraries. 5′ primers corresponded to vectorsequence that flanked cDNA inserts in the library being used astemplate, while 3′ primers corresponded to known sequence (EST or newlyderived 5′ PME-1 sequence). Southern Blotting using an end-labeled 20 bpoligonucleotide corresponding to known PME-1 sequence upstream of the 3′PCR primer was employed to identify authentic PME-1 products after eachreaction. PCR products containing 5′ extensions of the PME-1 sequencewere purified using a PCR product purification kit (Boehringer-Mannheim,Indianapolis, Ind.), cloned, and sequenced. New primers were designedfor PCR and Southern Blotting and then the above steps were repeateduntil the sequence of the remainder of the PME-1 coding region and aportion of the 5′ UTR were obtained.

Total mRNA was purified from HeLa cells using Trizol Reagent (LifeTechnologies, Gaithersburg, Md.) according to the manufacturer'sinstructions. RT-PCR was employed to obtain a PME-1 cDNA from HeLa cellmRNA. First strand synthesis was performed with Avian MyeloblastosisVirus reverse transcriptase (Boehringer-Mannheim, Indianapolis, Ind.) bythe manufacturer's protocol using a primer from the PME-1 3′ UTR(TGTTGAGGAGGGGTGGACAG) (SEQ ID NO: 1). Using pfu polymerase (Stratagene,La Jolla, Calif.), the product was used for PCR with the same 3′ primerand a primer from the PME-1 5′ UTR (TGTATGGGGACCTTCCTCCT) (SEQ ID NO:2)to generate a cDNA containing the entire PME-1 coding region and much ofthe 5′ UTR, including the in frame stop codon upstream of the putativestart ATG.

Obtaining the entire human PME-1 coding sequence required hundreds ofPCR reactions, scores of oligonucleotide primers, many Southern blotsand numerous subclonings and sequencing reactions. Most libraries didnot contain cDNAs with a full length PME-1 coding sequence.

Example 11 Purification of His-tagged PME-1 from bacteria expressingrecombinant His-tagged PME-1

E. coli (PR13Q) expressing recombinant His-tagged PME-1 from anisopropylthiogalactoside (IPTG) inducible lac promoter were grown to anO.D. at 600 nm of 0.7 and then induced with IPTG for 2-3 h. The PME-1coding sequence is fused in frame in a vector such as pThioHis A, B, C,pTrcHis A, B, C or pTrcHis 2A, B, C (Invitrogen, Carlsbad, Calif.).Cells were collected by centrifugation and broken open in the presenceof protease inhibitor by sonication or use of a French Pressure cell,using a lysis buffer containing normal saline (137 mM), and 20 mMTrisHCl (pH8.0). Lysates were cleared by centrifugation at ≧13,000×g,and supernatants were incubated in batch with a nickel-agarose columnmatrix (Chelating Sepharose, Pharmacia, Piscataway, N.J.) for 1-2 h at4° C. with rocking. Alternatively, a packed nickel-agarose column wasused and the supernatant was passed over it slowly several times. Ineither case the nickel-agarose/6XHis-PME-1 complexes were washed andthen His-tagged PME-1 was eluted with increasing amounts of imidizole(either with a step or continuous gradient). PME-1 protein thus isolatedwas dialyzed to remove the imidizole or analyzed on a Mono-Q column.Milligram amounts of PME-1 protein have been obtained from a liter ofculture.

Example 12 Assay for PP2A methylesterase activity

Epitope-tagged PP2A C subunits with ³H-methyl groups incorporated invitro were immunoprecipitated with anti-tag antibody and used assubstrate for PME-1. PME-1 enzyme sources assayed include: lysates ofbacteria or baculovirus-infected Sf9 insect cells expressing His-taggedPME-1 and immunoprecipitated PME-1 from baculovirus-infected Sf9 insectcells. Control lysates from bacteria or Sf9 cells not expressingrecombinant PME-1 were also incubated with tritiated substrate tomeasure non-specific background from the lysates. After 1 h incubationat 32° C., the amount of ³H-methyl groups remaining was assayed bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). PME-1 was clearlyable to demethylate PP2A C subunit as measured by this assay. TheHA-tagged PME-1 expressed in the baculovirus vector has beendemonstrated to have PP2A methylesterase activity.

In a second methylesterase assay, unlabeled epitope-tagged PP2A Csubunits were immunoprecipitated with anti-tag antibody and used assubstrate for PME-1. As PME-1 enzyme source, the following were used:cell lysates of bacteria expressing HA-tagged, His-tagged, and untaggedPME-1; cell lysates of HA-tagged or untagged PME-1-expressingbaculovirus-infected Sf9 insect cells; purified bacterial HA-taggedPME-1, purified (immunoprecipitated) baculovirus-infected Sf9 HA-taggedPME-1. Control lysates from bacteria or Sf9 cells not expressingrecombinant PME-1 were also incubated with substrate to measurenon-specific background from the lysates. After 1 h incubation at 32°C., the C subunit immunoprecipitates were washed and analyzed bySDS-PAGE. The proteins in the gels were electrophoretically transferredto nitrocellulose membranes and then the membranes were probed withmonoclonal antibody (made in our laboratory) that only recognizesunmethylated PP2A. A second probing of the same membrane with amethylation-insensitive antibody shows the actual amount of PP2A Csubunit in each lane. Comparison of the blotting signals for the twodifferent antibodies allow demethylation to be evaluated (the signal ofthe methylation-inhibited antibody gets stronger as PP2A C subunit isdemethylated). PME-1 was clearly able to demethylate PP2A C subunit, asmeasured by this assay.

An in vitro methylesterase activity assay using the PME-1 protein, forexample, produced as a recombinant human PME-1, can be used to screentest compounds for inhibition of PME-1. Inhibitors of PME-1 in e.g.,neoplastic cells slow the growth of those cells. Inhibitors could alsobe used to slow the growth in other hyperproliferative conditions. InAlzheimer's disease, PP2A has reduced activity. Identification ofcompounds which increase PP2A activity, for example, by appropriatelymodulating PME-1 activity, allows treatment to slow the progression ofAlzheimer's disease, and thus postpone loss of mental function inaffected patients.

Example 13 Computer analyses

The NCBI BLAST program [Altschul, S. F. et al. (1990) J. Mol. Biol.215:403-410] was used to probe various databases for PME-1 ESTs andrelated proteins. The DNASTAR Lasergene software package was utilizedfor alignments and identification of the PROSITE database lipase motiffound in PME-1.

Example 14 Northern Blot

Adult Balb/c mice were sacrificed and organs removed and flash-frozen inliquid nitrogen. total RNA from the organs was isolated using the RNeasykit (QIAGEN), and analyzed on formaldehyde-1% agarose gels to check forRNA integrity and to estimate the amount of the 18S and 28S RNAs. Basedon these estimates, similar amounts of RNA were separated onformaldehyde-1% agarose gels and transferred to GeneScreen nylonmembranes. After UV-crosslinking, the membranes were stained with a0.04% methylene blue solution to visualize the RNA. Filters were thenhybridized with a ³²P-radiolabeled probe generated by random primerlabeling of a DNA fragment from the 3′ untranslated region of the mousePME-1 cDNA. the probe, 395 bp in length, is an EcoRI-NotI fragment of aPME-1 EST clone (accession number W34856). The blots were used forautoradiography with X-ray film and/or analyzed on a STORMPhosphorimager (Molecular Dynamics, Sunnyvale, Calif.).

Example 15 Production of Polyclonal Antibodies Recognizing PME-1

Two different antisera recognizing PME-1 were raised in rabbits. Thefirst AR2, was raised against a 16-residue PME-1 peptide sequence(RIELAKTEKYWDGWFR) (amino acids 288-303, SEQ ID NO:5) found encoded inthe PME-1 cDNA. The peptide was conjugated to Keyhole Limpet Hemocyanin(KLH) via an added carboxy-terminal cysteine residue using a PieceImject conjugation kit, and the conjugate was used as immunogen. Thesecond antiserum, E37, was raised against a mixture of two-nickelagarose-purified, 6×His-tagged, bacterially expressed human PME-1fragments that together represent the carboxy-terminal half of theprotein. For each immunogen, a single female New Zealand white rabbitwas immunized and boosted multiple times using Freund's adjuvant.

Example 16 PME-1 Sequences from other organisms

Yeast PME-1 was found by homology searches of sequence databases usingthe NCBI BLAST program and the known human PME-1 sequence. Genomic yeastPME-1 sequence was examined and found to have no introns; therefore, wedesigned PCR primers for yeast PME-1 carried out PCR, cloned the PCRproduct into a bacterial vector and sequenced it to make sure no PCRerrors had occurred.

Table 3 shows the amino acid sequence of the yeast methylesterasehomolog of PME-1. Table 6 provides the coding sequence for the yeastPME-1 protein.

The C. elegans PME-1 coding sequence was deduced by homology withmammalian and yeast PME-1 sequenced. However, it should be noted thatthis gene product was not predicted by the Genefinder program.

Table 4 provides the amino acid sequence of the C. elegans PME-1 homologand Table 7 gives the coding sequence. Review of the EST sequencesrevealed two potential alternative splicing scenarios. The alternatewhich encoded an LLSTYCR amino acid segment (SEQ ID NO: 17) was ruledout based on the lack of a similar amino acid segment in the yeast PME-1protein and poor alignment with the human protein sequence.

Table 9 illustrates the alignment of human, C. elegans and S. cerevisiae(YHN5) PME-1 protein sequences. Residues identical with human PME-1 areas white-on-black. Residues corresponding to the Prosite motif forlipases employing an active site serine are boxed.

The mouse PME-1 sequences were found by search for EST sequences onGenBank with significant homology to the human PME-1 DNA sequencesdisclosed herein. Table 5 represents a portion of the mouse codingsequence generated by homology searches and computer-aided alignment ofthe mouse sequences to the human sequences and creating a consensussequence for the nucleotides of the various homologous ESTs. The first283 nucleotides of Table 5 are from a single EST (GenBank Accession No.AA555778). The next 465 nucleotides are given as X's because there wasno mouse sequence homologous to the corresponding human PME-1 cDNAsequence. It is understood that the actual length may not be exactly 465nucleotides. The following 527 nucleotides are from a single mouse EST(Accession No. AA644991.) The next seven nucleotides (1276-1282) arefrom an overlap of AA644991 with AA672810. The following 132 nucleotidesare from AA 672810 only. Then two other ESTs overlap; thus, most of theremaining nucleotides are quite certain, with the following exceptions.The nucleotides at positions 1942-1943 at somewhat ambiguous in that twoESTs have the identified sequence while others have TA, TN or T-. The Gat position 2167 is from 2 of 3 ESTs. R at 2169 is from a G and A in twoESTs. The sequence at 2174-2175 appears unreliable. Nucleotides2247-2270 are from a single EST (Accession No. EST AA260585) andnucleotides 2337-2409 are from a single minus-strand EST (Accession No.T25552).

Plants also have similar growth regulatoryphosphatase-kinase-methylation-demethylation systems, and there is aplant protein having significant homology to the mouse, human, yeast andnematode (C. elegans) PME-1 sequences, especially to the catalytic andGQMQGK (amino acids 333-338 of SEQ ID NO:5) regions of human PME-1. Theplant homolog(s) of PME-1 can be identified using techniques similar tothose described herein, including, but not limited to, the use ofsequence database searches in conjunction with PCR, RT-PCR and/orhybridization studies and immunological screening with antibodiesspecific for a PME-1 protein.

TABLE 1 H59Q and H118Q are catalytically inactive^(a) Csubunit-associated phosphatase activity (% wt)^(b) phosphorylase acdc2-phosphorylated C subunit (Means ± s.d.) Histone H1 (Mean ± s.d.)None 9 ± 2 2 ± 1 (vector only control) wt 100 100 H59Q 7 ± 1 2 ± 1 H118Q8 ± 3 2 ± 1 ^(a)PP2A activity present in anti-C subunit (HA tag)immunoprecipitates was measured using phosphorylase a andcdc2-phosphorylated histone H1 as substrates as described herein andnormalized to the wt value. The data represent four independentexperiments. Background phosphatase activity is probably due tonon-specific binding of a small amount of active, non epitope-tagged,endogenous C subunit.

TABLE 2 Nucleotide and Deduced Amino Acid Sequences for Human PME-1 (SEQID. NO:4 and SEQ ID NO:5 respectively)GGGCGTCGTTAGGGGAGCGAGTCGTGACCGGTTGGGCCACACTCAACGTGGGACGAAGCT 60TCGCCTACTGTTTGACTACGTGCGTGCAGCCTCCCCTCGATGTCGGCCCTCGAAAAGAGC 120                                      |{overscore (PME-1 coding sequence)}                                       |                                                             M  S  A  L  E  K  SATGCACCTCGGCCGCCTTCCCTCTCGCCCACCTCTACCCGGCAGCGGGGGCAGTCAGAGC 180                     PME-1 coding  sequence                   M  H  L  G  R  L  P  S  R  P  P  L  P  G  S  G  G  S  Q  S GGAGCCAAGATGCGAATGGGCCCTGGAAGAAAGCGGGACTTTTCCCCTGTTCCTTGGAGT 240                     PME-1 coding  sequence                   G  A  K  M  R  M  G  P  G  R  K  R  D  F   S   P  V  P  W  S CAGTATTTTGAGTCCATGGAAGATGTAGAAGTAGAGAATGAAACTGGCAAGGATACTTTT 300                     PME-1 coding  sequence                   Q  Y  F  E  S  M  E  D  V  E  V  E  N  E  T  G  K  D  T  F CGAGTCTACAAGAGTGGTTCAGAGGGTCCAGTCCTGCTCCTTCTGCATGGAGGAGGTCAT 360                     PME-1 coding  sequence                   R  V  Y  K  S  G  S  E  G  P  V  L  L  L  L  H  G  G  G  H TCTGCCCTTTCTTGGGCTGTGTTCACGGCAGCGATTATTAGTAGAGTTCAGTGTAGGATT 420                     PME-1 coding  sequence                   S  A  L  S  W  A  V  F  T  A  A  I  I  S  R  V  Q  C  R  I GTAGCTTTGGATCTGCGAAGTCATGGTGAAACAAAGGTCAAGAATCCTGAAGATCTGTCT 480                     PME-1 coding  sequence                   V  A  L  D  L  R  S  H  G  E  T  K  V  K  N  P  E  D  L  S GCAGAAACAATGGCAAAAGACGTTGGCAATGTGGTTGAAGCCATGTATGGGGACCTTCCT 540                     PME-1 coding  sequence                   A  E  T  M  A  K  D  V  G  N  V  V  E  A  M  Y  G  D  L  P CCTCCAATTATGCTGATTGGACATAGCATGGGTGGTGCTATTGCAGTCCACACAGCATCA 600                     PME-1 coding  sequence                   P  P  I  M  L  I  G  H  S  M  G  G  A  I  A  V  H  T  A  S TCCAACCTGGTACCAAGCCTCTTGGGTCTGTGCATGATTGATGTTGTAGAAGGTACAGCT 660                     PME-1 coding  sequence                   S  N  L  V  P  S  L  L  G  L  C  M  I  D  V  V  E  G  T  A ATGGATGCACTTAATAGCATGCAGAATTTCTTACGGGGTCGTCCTAAAACCTTCAAGTCT 720                     PME-1 coding  sequence                   M  D  A  L  N  S  M  Q  N  F  L  R  G  R  P  K  T  F  K  S CTGGAGAATGCTATTGAATGGAGTGTGAAGAGTGGCCAGATTCGAAATCTGGAGTCTGCC 780                     PME-1 coding  sequence                   L  E  N  A  I  E  W  S  V  K  S  G  Q  I  R  N  L  E  S  A CGTGTCTCAATGGTTGGCCAAGTCAAACAGTGTGAAGGAATTACAAGTCCAGAAGGCTCA 840                     PME-1 coding  sequence                   R  V  S  M  V  G  Q  V  K  Q  C  E  G  I  T  S  P  E  G  S AAATCTATAGTGGAAGGAATCATAGAGGAAGAAGAAGAAGATGAGGAAGGAAGTGAGTCT 900                     PME-1 coding  sequence                   K  S  I  V  E  G  I  I  E  E  E  E  E  D  E  E  G  S  E  S ATAAGCAAGAGGAAAAAGGAAGATGACATGGAGACCAAGAAAGACCATCCATACACCTGG 960                     PME-1 coding  sequence                   I  S  K  R  K  K  E  D  D  M  E  T  K  K  D  H  P  Y  T  W AGAATTGAACTGGCAAAAACAGAAAAATACTGGGACGGCTGGTTCCGAGGCTTATCCAAT 1020                     PME-1 coding  sequence                   R  I  E  L  A  K  T  E  K  Y  W  D  G  W  F  R  G  L  S  N CTCTTTCTTAGTTGTCCCATTCCTAAATTGCTGCTCTTGGCTGGTGTTGATAGATTGGAT 1080                     PME-1 coding  sequence                   L  F  L  S  C  P  I  P  K  L  L  L  L  A  G  V  D  R  L  D AAAGATCTGACCATTGGCCAGATGCAAGGGAAGTTCCAGATGCAGGTCCTACCCCAGTGT 1140                     PME-1 coding  sequence                   K  D  L  T  I  G  Q  M  Q  G  K  F  Q  M  Q  V  L  P  Q  C GGCCATGCAGTCCATGAGGATGCCCCTGACAAGGTAGCTGAAGCTGTTGCCACTTTCCTG 1200                     PME-1 coding  sequence                   G  H  A  V  H  E  D  A  P  D  K  V  A  E  A  V  A  T  F  L ATCCGGCACAGGTTTGCAGAACCCATCGGTGGATTCCAGTGTGTGTTTCCTGGCTGTTAG 1260                     PME-1 coding  sequence                   I  R  H  R  F  A  E  P  I  G  G  F  Q  C  V  F  P  G  C TGACCTGCTGTCCACCCCTCCTCAACATCGAGCTCTGTTGTAAATACGTCGCACCAGAGG 1320CCACTGTGATGCCACTGTCTCCTCTCCATCCCGCCCAGCCATGTGACACTGGCTCCCGGT 1380AGACGGGCACCCCGAGATGTACCAACCTTTTCATGTATTCTGCCAAAAGCATTGTTTTCC 1440AGGGCCCTTGACCAACATCGGCTTCCCCAGTCCAGGGCTCCCCTGCTCCTTTCCCTTCCC 1500TGTACTGGGGTAGCTCCTGCCTGCTCTCCCTGCGTTGCCTAGGGTAAAGCCTCCAGATTT 1560GCCATACTGAGCCCCTCTTCCTAGCATCAGGCGATACATCTGAGTTCAAATGTCTTCCCA 1620GGCTCAGGGACCTCCATTCCTTGAGATTGTCTTGGCATGGCCCAGCCCTGCCTCATGGGA 1680TGGACAATGCATGGGGTGGTCTTTATTTTTCCCTTTCAAATAAAACACTAGTCAGGTACC 1740GTTTTATCCCAGTCGTACTCTTCCAGGTTTGGAAGACCCAGAGAGGCCAAGATCCCATCC 1800TTAGCCATAGCGAGCGGTGGTGGTGGATAGCATCACAAGAAACGAGCCTGAAAATCAGGT 1860CCAGCCGGTCCAAGCACATGGCCTCCCATCTGGGAGAGCCCACTGTCCCACTCCCACATG 1920TCTGGGCACCTGCCCTGGGCTGAGGCCAGGCTGCTCCAGGGGCCTCCTGCGCCCTCACCT 1980GCCACAGAGCAACCCAGGTTAAATACAGCCCATGCACAAAGCCACAGGCCAAAGCCTATG 2040GAATTGTTTTTAATCATCAAATTTAACCATTTTCATAACTGGTTCCTGGAGGTGTGCAGT 2100GCCCCCTTGCCTCTTCAAACCTACAGCTTCTCTTTGCCATTTGTGGATTTCACATCACTC 2160CACACAGAAACATTACAGCCTGGCATCCCCAGTCTTTGCCTTCTTCCAGCTGCCTCGACA 2220CAGCACTGTGGCCTGTCCCTATTGCCCAGGCACGCCATTTCCAAGGGCAGGAAGGGGCAG 2280TGTCCTGAAGCCCATCTTTTCTGTGACTGTCTTAGGTGATGTGTAGCCCCCTCCACCTTT 2340CCACTCAACAACCTCCCACCCCTGTCCTGCTGCATGGTCCGGAGTCTGGGACCTACTTTG 2400TTTTTTGTTATTTATGACCTTGTTTAAAGAAAATAAATATGTCCCAACCTTTAAAAAAAA 2460AAAAAAAAAAAAAAAAAAAAAAAA 2484

TABLE 3 Saccharomyces cervisiae PME-1 Amino Acid Sequence (SEQ ID NO:6)MSDDLRRKIALSQFERAKNVLDATFQEAYEDDENDGDALGSLPSFNGQSNRNRKYTGKTGSTTDRISSKEKSSLPTWSDFFDNKELVSLPDRDLDVNTYYTLPTSLLSNTTSIPIFIFHHGAGSSGLSFANLAKELNTKLEGRCGCFAFDARGHAETKFKKADAPICFDRDSFIKDFVSLLNYWFKSKISQEPLQKVSVILIGHSLGGSICTFAYPKLSTELQKKILGITMLDIVEEAATMALNKVEHFLQNTPNVFESINDAVDWHVQHALSRLRSSAEIAIPALFAPLKSGKVVRITNLKTFSPFWDTWFTDLSHSFVGLPVSKLLILAGNENLDKELIVGQMQGKYQLVVFQDSGHFIQEDSPIKTAITLIDFWKRNDSRNVVIKTNWGQHK TVQNT

From GenBank sequence sequences identified as encoding a hypotheticalprotein; deposited by JOHNSTON M., ANDREWS S., BRINKMAN R., COOPER J.,DING H., DOVER J., DU Z., FAVELLO A., FULTON L., GATTUNG S., GEISEL C.,KIRSTEN J., KUCABA T., HILLIER, L., JIER M., JOHNSTON L., LANGSTON Y.,ATREILLE P., LOUIS E. J., MACRI C., MARDIS E., MENEZES S., MOUSER L.,NHAN M., RIFKIN L., RILES L., ST.PETER H., TREVASKIS E., VAUGHAN K.,VIGNATI D., WILCOX L., WOHLDMAN P., WATERSTON R., WILSON R., VAUDIN M.;See also SCIENCE 265:2077-2082(1994).

ACCESSION: P38796; PIR: S46814 #type complete ACCESSION: S46814 GB:YSCH9205 ACCESSION: U10556 DESC HYPOTHETICAL 44.9 KD PROTEIN INERG7-NMD2 INTERGENIC REGION. DATE FEB. 1, 1995 (REL. 31, CREATED) FEB.1, 1995 (REL. 31, LAST SEQUENCE UPDATE) FEB. 1, 1995 (REL. 31, LASTANNOTATION UPDATE) GENE YHR075C. #map_position 8R COM SEQUENCE FROM N.A.STRAIN=S288C/AB972; MED MEDLINE; 94378003. AUTH TAXONOMY EUKARYOTA;FUNGI; ASCOMYCOTINA; HEMIASCOMYCETES. COMMENT Nucleic Acid Featurestranslated to generate this entry: CDS complement(9569 . . . 10771)/codon_start=1/evidence=not_experimental/db_xref=“PID:g500835”

TABLE 4 Caenorhabditis elegans PME-1 Amino Acid Sequence. (SEQ ID NO:7)MSDDKLDTLPDLQSETSHVTTPHRQNDLLRQAVTHGRPPPVPSTSTSGKKREMSELPWSDFFDEKKDANIDGDVFNVYIKGNEGPIFYLLHGGGYSGLTWACFAKELATLISCRVVAPDLRGHGDTKCSDEHDLSKETQIKDIGAIFKNIFGEDDSPVCIVGHSMGGALAIHTLNAKMISSKVAALIVIDVVEGSAMEALGGMVHFLHSRPSSFPSIEKAIHWCLSSGTARNPTAARVSMPSQIREVSEHEYTWRIDLTTTEQYWKGWFEGLSKEFLGCSVPKMLVLAGVDRLDRDLTIGQMQGKFQTCVLPKVGHCVQEDSPQNLADEVGRFACRHRIAQPKFSALASPPDPAILEYRKRIHHQ

TABLE 5 Partial CDNA Sequence of Mus musculus PME-1 homolog (SEQ IDNO:8) TTGTACTGCACGTATCGTGGGACGGACCTTGGGCCACTGTTGTCGACGTGCGGCCTCCCTTTGATGTCGGCCCTTGAAAAAAGCATGCACCTCGGCCGCCTACCTTCTCGCCCTCCTCTACCCGGCAGCGGGGGCAGTCAGAGCGGACGCAAGATGCGGATGGGCCCTGGACGGAAGCGGGACTTTACCCCTGTCCCATGGAGTCAGTACTTTGAGTCAATGGAAGATGTGGAAGTGGAGAATGAAACTGGCAAGGATACTTTTCGAGTTTACAAGATTGGTTXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTTCGGATCCTTGGCCAAGTCAAACAGTGTGAAGGAATTACAAGTCCAGAAGGTTCCAAATCCATAGTGGAAGGAATCATAGAGGAGGAGGAAGAAGATGAGGAAGGAAGTGAGTCAGTTAACAAGAGGAAAAAGGAAGACGACATGGAAACCAAGAAGGATCACCCATACACCTGGAGAATTGAGCTGGCAAAAACAGAAAAGTACTGGGATGGCTGGTTCCGGGGCTTATCCAATCTCTTTCTTAGCTGTCCTATTCCTAAACTGCTGCTCTTGGCGGGTGTTGACAGATTGGATAAAGATCTGACCATAGGCCAGATGCAGGGGAAGTTCCAGATGCAGGTCTTACCCCAGTGTGGCCATGCAGTCCATGAGGATGCCCCTGACAAGGTAGCTGAAGCTGTTGCCACTTTCCTGATCCGGCACAGGTTTGCAGAGCCCATCGGAGGATTCCAGTGTGTGTTTACTGGCTGCTAGTGACCTGCTGTCTACTCCTCCCTCTACATTGAGCTCTGTTGTAAATACATCGCACCAGAGGCCACTGTGACGCCGCTGTCTCCTCCTCTCCATCCCGCCCAGCCATGTGACACCGGCTCTTGTAGAGGGCATCCCCAGATGTCCAAACCCTTTCCTGTGTACTGTTGAAAGCATTGTTCTTCAGGGCCCTTGTCCAACAGTGGCCCGTGCAGTCTGGGGTCCACAGCTCTTCCTCTCCTTCCTGTGCTCCCTGCCTTGCCTAGGATGAAGCCTCCAGCGCTGCTCCCTGGCCCTGTTCCTGGCATATGGCAATGTACCCCAGGCTCAGGGATCTCCCTTCCTTGAGGATGTTCTTGGCATGGTCCTGCCCTACCTCATGGGATGGGCAATGCACACACTGGCCCTTATTTTTCCCTTTCAAATAAAACACCAGTCAGGTACCTTTATCCCAGTCTTAACTGTCCCAAATCTGGAAGGTCCAGAGTAAGCAGGATTCAGGGAGAGGGAGTGGATAGCAAGTATCCCAAGAAACCAACCTGTAAGTCAGGTCCAGCCAGTCCAAGCACATGGCTTCCCATCTGGGTGAGCCCACTGTCCCACTCCCACATGTCTGGGCACCTGCCCTGGGCTGAGGCCAGGCTGCTCCAAGGGCCGCATGAGCCCTAATCTGCCACAGAGCAACCCAGGTTAAACACAGCCCATGCACAAAGCCACAGGCTAAATCCTGTGGAATTGTTTTTAATGACTGAATTTAACCATTTTCATAGTTGGTTCCTGGAGGTGTGCCAAGTGCCCGCTTGCCTCTTCTAGACCCACAGCTTCTTGATCCACTTGTGGTTTCCATGTCACTAATGTAGAAACATCATGGACTAGCATCCCCAGTCTTTGCCCTCATCCAGCTGTCGCAGCGCACACTGGGGCCTCCCCCTGCTGCCCAGGGGGGGRCGGGGTGGGCAGCCTCCTGAAACCCATCTTTCTGTGACTGTCTTAGGTGACGTGTAGCCCTCTTCCGTTTTTTCACCCAACAACTTCCTCTGTCCTGCTGCACGGTCCAGAGTCTGGGACCGACTTTGTTTCTTTGTTATTTATGATCTTGTTTAAAGAAAATAAATATCTCCCAACCTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA A

TABLE 6 S. cerevisiae PME-1 Coding Sequence (SEQ ID NO:9)ATGTCTGACGATTTGAGAAGAAAAATTGCTTTATCCCAGTTTGAGAGAGCCAAGAATGTTCTAGATGCGACATTCCAAGAAGCATACGAGGATGATGAAAACGATGGTGATGCATTAGGTTCCCTGCCATCATTTAATGGACAATCAATAGGAACAGAAAATATACGGGCAAAACCGGTAGTACTACTGATAGAATTTCAAGTAAGGAAAAGAGTAGTTTACCCACTTGGAGTGATTTTTTTGATAATAAGGAGTTGGTAAGTCTTCCTGATAGAGATCTGGACGTAAATACATACTATACATTACCTACTTCATTGTTATCAAATACCACTTCAATTCCCATCTTTATTTTCCACCATGGGGCGGGCTCCTCAGGTTTATCATTTGCAAACTTGGCCAAGGAATTAAATACTAAACTAGAAGGAAGATGCGGATGCTTTGCATTTGATGCTAGGGGGCATGCAGAAACAAAGTTTAAGAAGGCTGATGCGCCTATATGCTTTGACAGGGACTCTTTTATCAAAGATTTTGTAAGCCTGCTAAATTATTGGTTTAAGTCTAAAATAAGCCAAGAGCCACTTCAGAAGGTATCTGTTATACTAATTGGTCATTCCCTTGGTGGAAGTATATGTACTTTTGCGTACCCTAAATTATCAACAGAACTACAAAAGAAAATTCTTGGTATTACTATGTTAGATATTGTAGAAGAGGCTGCCATTATGGCCTTAAATAAAGTTGAACATTTTTTGCAGAATACACCCAATGTATTTGAATCAATTAATGACGCTGTCGATTGGCACGTTCAACACGCGTTATCGAGATTGAGGTCAAGCGCCGAAATTGCTATACCAGCTTTATTTGCTCCGCTCAAGTCAGGGAAAGTTGTCAGGATAACAAACCTTAAGACCTTTAGCCCTTTCTGGGACACATGGTTTACCGATCTGTCGCACTCCTTTGTTGGCTTACCTGTTAGTAAATTATTAATATTGGCGGGAAACGAAAATCTCGATAAAGAATTAATTGTGGGGCAAATGCAAGGTAAATATCAGTTGGTAGTTTTCCAAGATTCCGGGCATTTCATTCAAGAAGATTCGCCTATAAAAACAGCAATCACTTTAATTGATTTCTGGAAGCGGAACGATTCTAGGAATGTAGTAATCAAGACTAATTGGGGTCAACACAAAACCGT GCAAAATACATAA

TABLE 7 C. elegans PME-1 Coding Sequence (SEQ ID NO:10)ATGTCCGACGATAAATTAGACACTCTTCCGGATCTTCAATCGGAAACGTCACATGTCACAACTCCTCACAGGCAAAATGATCTTCTTCGTCAAGCGGTCACTCATGGAAGGCCACCACCAGTTCCGAGCACATCAACTTCTGGAAAGAAACGAGAAATGTCTGAACTACCGTGGTCAGATTTTTTTGATGAAAAGAAGGACGCAAACATTGATGGAGATGTTTTCAATGTGTACATAAAGGGAAATGAAGGTCCAATTTTCTATTTGCTTCACGGTGGAGGTTATTCAGGCCTCACATGGGCGTGTTTTGCGAAAGAATTGGCAACTTTAATATCATGCAGAGTTGTTGCACCTGATTTAAGAGGACACGGCGACACTAAATGTTCTGATGAGCACGATCTTTCGAAAGAAACCCAAATAAAGGATATTGGAGCAATCTTCAAGAACATTTTCGGCGAAGACGATTCACCAGTATGCATTGTTGGACACAGTATGGGTGGTGCATTGGCCATTCATACATTGAATGCAAAGATGATTTCTTCAAAAGTCGCTGCACTCATTGTCATTGATGTTGTCGAAGGTTCCGCTATGGAAGCACTTGGAGGAATGGTTCATTTTTTACATTCAAGGCCTTCTTCATTTCCTTCTATCGAAAAAGCCATTCACTGGTGCCTTTCTTCGGGTACAGCGAGGAATCCCACAGCTGCACGGGTCTCAATGCCGTCTCAAATTAGAGAAGTATCGGAACACGAGTACACTTGGCGAATTGATTTAACAACAACAGAACAGTACTGGAAAGGATGGTTTGAAGGATTATCCAAAGAATTTTTGGGATGTTCCGTTCCGAAGATGCTTGTTCTAGCGGGCGTTGATCGGCTGGACAGGGATCTCACAATTGGTCAAATGCAGGGAAAGTTTCAGACTTGTGTGTTACCAAAAGTTGGACATTGTGTTCAGGAAGATAGCCCACAAAATCTTGCAGATGAAGTCGGAAGATTCGCTTGCCGCCATAGAATTGCCCAACCGAAATTCTCAGCCCTTGCATCACCACCAGATCCAGCGATTCTCGAATACAGAA AACGTCATCACCAATAA

TABLE 8 Comparison of the sequences surrounding the putative or knownactive site serines of PME-1 proteins and CheB Species First residue shSequence SEQ ID NO: Human PME-1 150 IMLIGHSMG 11 C. elegans PME-1 158VCIVGHSMG 12 S. cerevisiae PME-1 199 VILIGHSLG 13 S. typhimurium CheB158 LIAIGASTG 14 The PME-1 and CheB residues matching the signaturemotif ([LIV]-x-[LIVFY]-[LIVST]-G-[HYWV]-S-x-G-[GSTAC]) (SEQ ID NO:15)for lipases utilizing an active site serine are underlined. The serinepresent in each sequence is the predicted (PME-1) or known (CheB) activesite serine.

TABLE 9 Alignment of human, C. elegans and S. cerevisiae PME-1 proteinsequences (SEQ ID NO:5, SEQ ID NO:7, and SEQ ID:6 respectively)

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 17 <210> SEQ ID NO 1 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:      oligonucleotide <400> SEQUENCE: 1tgttgaggag gggtggacag             #                  #                   # 20 <210> SEQ ID NO 2 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:      oligonucleotide <400> SEQUENCE: 2tgtatgggga ccttcctcct             #                  #                   # 20 <210> SEQ ID NO 3 <211> LENGTH: 16<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3Arg Ile Glu Leu Ala Lys Thr Glu Lys Tyr Tr #p Asp Gly Trp Phe Arg  1               5  #                 10  #                 15<210> SEQ ID NO 4 <211> LENGTH: 2484 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (100)..(1257) <400> SEQUENCE: 4gggcgtcgtt aggggagcga gtcgtgaccg gttgggccac actcaacgtg gg#acgaagct     60 tcgcctactg tttgactacg tgcgtgcagc ctcccctcg atg tcg gcc# ctc gaa       114                    #                  #       Met Ser Ala Leu Glu                    #                  #         1          #      5 aag agc atg cac ctc ggc cgc ctt ccc tct cg#c cca cct cta ccc ggc      162Lys Ser Met His Leu Gly Arg Leu Pro Ser Ar #g Pro Pro Leu Pro Gly                 10  #                 15  #                 20agc ggg ggc agt cag agc gga gcc aag atg cg#a atg ggc cct gga aga      210Ser Gly Gly Ser Gln Ser Gly Ala Lys Met Ar #g Met Gly Pro Gly Arg             25      #             30      #             35aag cgg gac ttt tcc cct gtt cct tgg agt ca#g tat ttt gag tcc atg      258Lys Arg Asp Phe Ser Pro Val Pro Trp Ser Gl #n Tyr Phe Glu Ser Met         40          #         45          #         50gaa gat gta gaa gta gag aat gaa act ggc aa#g gat act ttt cga gtc      306Glu Asp Val Glu Val Glu Asn Glu Thr Gly Ly #s Asp Thr Phe Arg Val     55              #     60              #     65tac aag agt ggt tca gag ggt cca gtc ctg ct#c ctt ctg cat gga gga      354Tyr Lys Ser Gly Ser Glu Gly Pro Val Leu Le #u Leu Leu His Gly Gly 70                  # 75                  # 80                  # 85ggt cat tct gcc ctt tct tgg gct gtg ttc ac#g gca gcg att att agt      402Gly His Ser Ala Leu Ser Trp Ala Val Phe Th #r Ala Ala Ile Ile Ser                 90  #                 95  #                100aga gtt cag tgt agg att gta gct ttg gat ct#g cga agt cat ggt gaa      450Arg Val Gln Cys Arg Ile Val Ala Leu Asp Le #u Arg Ser His Gly Glu            105       #           110       #           115aca aag gtc aag aat cct gaa gat ctg tct gc#a gaa aca atg gca aaa      498Thr Lys Val Lys Asn Pro Glu Asp Leu Ser Al #a Glu Thr Met Ala Lys        120           #       125           #       130gac gtt ggc aat gtg gtt gaa gcc atg tat gg#g gac ctt cct cct cca      546Asp Val Gly Asn Val Val Glu Ala Met Tyr Gl #y Asp Leu Pro Pro Pro    135               #   140               #   145att atg ctg att gga cat agc atg ggt ggt gc#t att gca gtc cac aca      594Ile Met Leu Ile Gly His Ser Met Gly Gly Al #a Ile Ala Val His Thr150                 1 #55                 1 #60                 1 #65gca tca tcc aac ctg gta cca agc ctc ttg gg#t ctg tgc atg att gat      642Ala Ser Ser Asn Leu Val Pro Ser Leu Leu Gl #y Leu Cys Met Ile Asp                170   #               175   #               180gtt gta gaa ggt aca gct atg gat gca ctt aa#t agc atg cag aat ttc      690Val Val Glu Gly Thr Ala Met Asp Ala Leu As #n Ser Met Gln Asn Phe            185       #           190       #           195tta cgg ggt cgt cct aaa acc ttc aag tct ct#g gag aat gct att gaa      738Leu Arg Gly Arg Pro Lys Thr Phe Lys Ser Le #u Glu Asn Ala Ile Glu        200           #       205           #       210tgg agt gtg aag agt ggc cag att cga aat ct#g gag tct gcc cgt gtc      786Trp Ser Val Lys Ser Gly Gln Ile Arg Asn Le #u Glu Ser Ala Arg Val    215               #   220               #   225tca atg gtt ggc caa gtc aaa cag tgt gaa gg#a att aca agt cca gaa      834Ser Met Val Gly Gln Val Lys Gln Cys Glu Gl #y Ile Thr Ser Pro Glu230                 2 #35                 2 #40                 2 #45ggc tca aaa tct ata gtg gaa gga atc ata ga#g gaa gaa gaa gaa gat      882Gly Ser Lys Ser Ile Val Glu Gly Ile Ile Gl #u Glu Glu Glu Glu Asp                250   #               255   #               260gag gaa gga agt gag tct ata agc aag agg aa#a aag gaa gat gac atg      930Glu Glu Gly Ser Glu Ser Ile Ser Lys Arg Ly #s Lys Glu Asp Asp Met            265       #           270       #           275gag acc aag aaa gac cat cca tac acc tgg ag#a att gaa ctg gca aaa      978Glu Thr Lys Lys Asp His Pro Tyr Thr Trp Ar #g Ile Glu Leu Ala Lys        280           #       285           #       290aca gaa aaa tac tgg gac ggc tgg ttc cga gg#c tta tcc aat ctc ttt     1026Thr Glu Lys Tyr Trp Asp Gly Trp Phe Arg Gl #y Leu Ser Asn Leu Phe    295               #   300               #   305ctt agt tgt ccc att cct aaa ttg ctg ctc tt#g gct ggt gtt gat aga     1074Leu Ser Cys Pro Ile Pro Lys Leu Leu Leu Le #u Ala Gly Val Asp Arg310                 3 #15                 3 #20                 3 #25ttg gat aaa gat ctg acc att ggc cag atg ca#a ggg aag ttc cag atg     1122Leu Asp Lys Asp Leu Thr Ile Gly Gln Met Gl #n Gly Lys Phe Gln Met                330   #               335   #               340cag gtc cta ccc cag tgt ggc cat gca gtc ca#t gag gat gcc cct gac     1170Gln Val Leu Pro Gln Cys Gly His Ala Val Hi #s Glu Asp Ala Pro Asp            345       #           350       #           355aag gta gct gaa gct gtt gcc act ttc ctg at#c cgg cac agg ttt gca     1218Lys Val Ala Glu Ala Val Ala Thr Phe Leu Il #e Arg His Arg Phe Ala        360           #       365           #       370gaa ccc atc ggt gga ttc cag tgt gtg ttt cc#t ggc tgt tagtgacctg      1267Glu Pro Ile Gly Gly Phe Gln Cys Val Phe Pr #o Gly Cys    375               #   380               #   385ctgtccaccc ctcctcaaca tcgagctctg ttgtaaatac gtcgcaccag ag#gccactgt   1327gatgccactg tctcctctcc atcccgccca gccatgtgac actggctccc gg#tagacggg   1387caccccgaga tgtaccaacc ttttcatgta ttctgccaaa agcattgttt tc#cagggccc   1447ttgaccaaca tcggcttccc cagtccaggg ctcccctgct cctttccctt cc#ctgtactg   1507gggtagctcc tgcctgctct ccctgcgttg cctagggtaa agcctccaga tt#tgccatac   1567tgagcccctc ttcctagcat caggcgatac atctgagttc aaatgtcttc cc#aggctcag   1627ggacctccat tccttgagat tgtcttggca tggcccagcc ctgcctcatg gg#atggacaa   1687tgcatggggt ggtctttatt tttccctttc aaataaaaca ctagtcaggt ac#cgttttat   1747cccagtcgta ctcttccagg tttggaagac ccagagaggc caagatccca tc#cttagcca   1807tagcgagcgg tggtggtgga tagcatcaca agaaacgagc ctgaaaatca gg#tccagccg   1867gtccaagcac atggcctccc atctgggaga gcccactgtc ccactcccac at#gtctgggc   1927acctgccctg ggctgaggcc aggctgctcc aggggcctcc tgcgccctca cc#tgccacag   1987agcaacccag gttaaataca gcccatgcac aaagccacag gccaaagcct at#ggaattgt   2047ttttaatcat caaatttaac cattttcata actggttcct ggaggtgtgc ag#tgccccct   2107tgcctcttca aacctacagc ttctctttgc catttgtgga tttcacatca ct#ccacacag   2167aaacattaca gcctggcatc cccagtcttt gccttcttcc agctgcctcg ac#acagcact   2227gtggcctgtc cctattgccc aggcacgcca tttccaaggg caggaagggg ca#gtgtcctg   2287aagcccatct tttctgtgac tgtcttaggt gatgtgtagc cccctccacc tt#tccactca   2347acaacctccc acccctgtcc tgctgcatgg tccggagtct gggacctact tt#gttttttg   2407ttatttatga ccttgtttaa agaaaataaa tatctcccaa cctttaaaaa aa#aaaaaaaa   2467 aaaaaaaaaa aaaaaaa              #                  #                   # 2484 <210> SEQ ID NO 5 <211> LENGTH: 386<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 5Met Ser Ala Leu Glu Lys Ser Met His Leu Gl #y Arg Leu Pro Ser Arg  1               5  #                 10  #                 15Pro Pro Leu Pro Gly Ser Gly Gly Ser Gln Se #r Gly Ala Lys Met Arg             20      #             25      #             30Met Gly Pro Gly Arg Lys Arg Asp Phe Ser Pr #o Val Pro Trp Ser Gln         35          #         40          #         45Tyr Phe Glu Ser Met Glu Asp Val Glu Val Gl #u Asn Glu Thr Gly Lys     50              #     55              #     60Asp Thr Phe Arg Val Tyr Lys Ser Gly Ser Gl #u Gly Pro Val Leu Leu 65                  # 70                  # 75                  # 80Leu Leu His Gly Gly Gly His Ser Ala Leu Se #r Trp Ala Val Phe Thr                 85  #                 90  #                 95Ala Ala Ile Ile Ser Arg Val Gln Cys Arg Il #e Val Ala Leu Asp Leu            100       #           105       #           110Arg Ser His Gly Glu Thr Lys Val Lys Asn Pr #o Glu Asp Leu Ser Ala        115           #       120           #       125Glu Thr Met Ala Lys Asp Val Gly Asn Val Va #l Glu Ala Met Tyr Gly    130               #   135               #   140Asp Leu Pro Pro Pro Ile Met Leu Ile Gly Hi #s Ser Met Gly Gly Ala145                 1 #50                 1 #55                 1 #60Ile Ala Val His Thr Ala Ser Ser Asn Leu Va #l Pro Ser Leu Leu Gly                165   #               170   #               175Leu Cys Met Ile Asp Val Val Glu Gly Thr Al #a Met Asp Ala Leu Asn            180       #           185       #           190Ser Met Gln Asn Phe Leu Arg Gly Arg Pro Ly #s Thr Phe Lys Ser Leu        195           #       200           #       205Glu Asn Ala Ile Glu Trp Ser Val Lys Ser Gl #y Gln Ile Arg Asn Leu    210               #   215               #   220Glu Ser Ala Arg Val Ser Met Val Gly Gln Va #l Lys Gln Cys Glu Gly225                 2 #30                 2 #35                 2 #40Ile Thr Ser Pro Glu Gly Ser Lys Ser Ile Va #l Glu Gly Ile Ile Glu                245   #               250   #               255Glu Glu Glu Glu Asp Glu Glu Gly Ser Glu Se #r Ile Ser Lys Arg Lys            260       #           265       #           270Lys Glu Asp Asp Met Glu Thr Lys Lys Asp Hi #s Pro Tyr Thr Trp Arg        275           #       280           #       285Ile Glu Leu Ala Lys Thr Glu Lys Tyr Trp As #p Gly Trp Phe Arg Gly    290               #   295               #   300Leu Ser Asn Leu Phe Leu Ser Cys Pro Ile Pr #o Lys Leu Leu Leu Leu305                 3 #10                 3 #15                 3 #20Ala Gly Val Asp Arg Leu Asp Lys Asp Leu Th #r Ile Gly Gln Met Gln                325   #               330   #               335Gly Lys Phe Gln Met Gln Val Leu Pro Gln Cy #s Gly His Ala Val His            340       #           345       #           350Glu Asp Ala Pro Asp Lys Val Ala Glu Ala Va #l Ala Thr Phe Leu Ile        355           #       360           #       365Arg His Arg Phe Ala Glu Pro Ile Gly Gly Ph #e Gln Cys Val Phe Pro    370               #   375               #   380 Gly Cys 385<210> SEQ ID NO 6 <211> LENGTH: 400 <212> TYPE: PRT<213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 6Met Ser Asp Asp Leu Arg Arg Lys Ile Ala Le #u Ser Gln Phe Glu Arg  1               5  #                 10  #                 15Ala Lys Asn Val Leu Asp Ala Thr Phe Gln Gl #u Ala Tyr Glu Asp Asp             20      #             25      #             30Glu Asn Asp Gly Asp Ala Leu Gly Ser Leu Pr #o Ser Phe Asn Gly Gln         35          #         40          #         45Ser Asn Arg Asn Arg Lys Tyr Thr Gly Lys Th #r Gly Ser Thr Thr Asp     50              #     55              #     60Arg Ile Ser Ser Lys Glu Lys Ser Ser Leu Pr #o Thr Trp Ser Asp Phe 65                  # 70                  # 75                  # 80Phe Asp Asn Lys Glu Leu Val Ser Leu Pro As #p Arg Asp Leu Asp Val                 85  #                 90  #                 95Asn Thr Tyr Tyr Thr Leu Pro Thr Ser Leu Le #u Ser Asn Thr Thr Ser            100       #           105       #           110Ile Pro Ile Phe Ile Phe His His Gly Ala Gl #y Ser Ser Gly Leu Ser        115           #       120           #       125Phe Ala Asn Leu Ala Lys Glu Leu Asn Thr Ly #s Leu Glu Gly Arg Cys    130               #   135               #   140Gly Cys Phe Ala Phe Asp Ala Arg Gly His Al #a Glu Thr Lys Phe Lys145                 1 #50                 1 #55                 1 #60Lys Ala Asp Ala Pro Ile Cys Phe Asp Arg As #p Ser Phe Ile Lys Asp                165   #               170   #               175Phe Val Ser Leu Leu Asn Tyr Trp Phe Lys Se #r Lys Ile Ser Gln Glu            180       #           185       #           190Pro Leu Gln Lys Val Ser Val Ile Leu Ile Gl #y His Ser Leu Gly Gly        195           #       200           #       205Ser Ile Cys Thr Phe Ala Tyr Pro Lys Leu Se #r Thr Glu Leu Gln Lys    210               #   215               #   220Lys Ile Leu Gly Ile Thr Met Leu Asp Ile Va #l Glu Glu Ala Ala Ile225                 2 #30                 2 #35                 2 #40Met Ala Leu Asn Lys Val Glu His Phe Leu Gl #n Asn Thr Pro Asn Val                245   #               250   #               255Phe Glu Ser Ile Asn Asp Ala Val Asp Trp Hi #s Val Gln His Ala Leu            260       #           265       #           270Ser Arg Leu Arg Ser Ser Ala Glu Ile Ala Il #e Pro Ala Leu Phe Ala        275           #       280           #       285Pro Leu Lys Ser Gly Lys Val Val Arg Ile Th #r Asn Leu Lys Thr Phe    290               #   295               #   300Ser Pro Phe Trp Asp Thr Trp Phe Thr Asp Le #u Ser His Ser Phe Val305                 3 #10                 3 #15                 3 #20Gly Leu Pro Val Ser Lys Leu Leu Ile Leu Al #a Gly Asn Glu Asn Leu                325   #               330   #               335Asp Lys Glu Leu Ile Val Gly Gln Met Gln Gl #y Lys Tyr Gln Leu Val            340       #           345       #           350Val Phe Gln Asp Ser Gly His Phe Ile Gln Gl #u Asp Ser Pro Ile Lys        355           #       360           #       365Thr Ala Ile Thr Leu Ile Asp Phe Trp Lys Ar #g Asn Asp Ser Arg Asn    370               #   375               #   380Val Val Ile Lys Thr Asn Trp Gly Gln His Ly #s Thr Val Gln Asn Thr385                 3 #90                 3 #95                 4 #00<210> SEQ ID NO 7 <211> LENGTH: 364 <212> TYPE: PRT<213> ORGANISM: Caenorhabditis elegans <400> SEQUENCE: 7Met Ser Asp Asp Lys Leu Asp Thr Leu Pro As #p Leu Gln Ser Glu Thr  1               5  #                 10  #                 15Ser His Val Thr Thr Pro His Arg Gln Asn As #p Leu Leu Arg Gln Ala             20      #             25      #             30Val Thr His Gly Arg Pro Pro Pro Val Pro Se #r Thr Ser Thr Ser Gly         35          #         40          #         45Lys Lys Arg Glu Met Ser Glu Leu Pro Trp Se #r Asp Phe Phe Asp Glu     50              #     55              #     60Lys Lys Asp Ala Asn Ile Asp Gly Asp Val Ph #e Asn Val Tyr Ile Lys 65                  # 70                  # 75                  # 80Gly Asn Glu Gly Pro Ile Phe Tyr Leu Leu Hi #s Gly Gly Gly Tyr Ser                 85  #                 90  #                 95Gly Leu Thr Trp Ala Cys Phe Ala Lys Glu Le #u Ala Thr Leu Ile Ser            100       #           105       #           110Cys Arg Val Val Ala Pro Asp Leu Arg Gly Hi #s Gly Asp Thr Lys Cys        115           #       120           #       125Ser Asp Glu His Asp Leu Ser Lys Glu Thr Gl #n Ile Lys Asp Ile Gly    130               #   135               #   140Ala Ile Phe Lys Asn Ile Phe Gly Glu Asp As #p Ser Pro Val Cys Ile145                 1 #50                 1 #55                 1 #60Val Gly His Ser Met Gly Gly Ala Leu Ala Il #e His Thr Leu Asn Ala                165   #               170   #               175Lys Met Ile Ser Ser Lys Val Ala Ala Leu Il #e Val Ile Asp Val Val            180       #           185       #           190Glu Gly Ser Ala Met Glu Ala Leu Gly Gly Me #t Val His Phe Leu His        195           #       200           #       205Ser Arg Pro Ser Ser Phe Pro Ser Ile Glu Ly #s Ala Ile His Trp Cys    210               #   215               #   220Leu Ser Ser Gly Thr Ala Arg Asn Pro Thr Al #a Ala Arg Val Ser Met225                 2 #30                 2 #35                 2 #40Pro Ser Gln Ile Arg Glu Val Ser Glu His Gl #u Tyr Thr Trp Arg Ile                245   #               250   #               255Asp Leu Thr Thr Thr Glu Gln Tyr Trp Lys Gl #y Trp Phe Glu Gly Leu            260       #           265       #           270Ser Lys Glu Phe Leu Gly Cys Ser Val Pro Ly #s Met Leu Val Leu Ala        275           #       280           #       285Gly Val Asp Arg Leu Asp Arg Asp Leu Thr Il #e Gly Gln Met Gln Gly    290               #   295               #   300Lys Phe Gln Thr Cys Val Leu Pro Lys Val Gl #y His Cys Val Gln Glu305                 3 #10                 3 #15                 3 #20Asp Ser Pro Gln Asn Leu Ala Asp Glu Val Gl #y Arg Phe Ala Cys Arg                325   #               330   #               335His Arg Ile Ala Gln Pro Lys Phe Ser Ala Le #u Ala Ser Pro Pro Asp            340       #           345       #           350Pro Ala Ile Leu Glu Tyr Arg Lys Arg His Hi #s Gln         355          #       360 <210> SEQ ID NO 8 <211> LENGTH: 2409 <212> TYPE: DNA<213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (1)..(2409) <223> OTHER INFORMATION: N is A, T, G or #C. <400> SEQUENCE: 8ttgtactgca cgtatcgtgg gacggacctt gggccactgt tgtcgacgtg cg#gcctccct     60ttgatgtcgg cccttgaaaa aagcatgcac ctcggccgcc taccttctcg cc#ctcctcta    120cccggcagcg ggggcagtca gagcggacgc aagatgcgga tgggccctgg ac#ggaagcgg    180gactttaccc ctgtcccatg gagtcagtac tttgagtcaa tggaagatgt gg#aagtggag    240aatgaaactg gcaaggatac ttttcgagtt tacaagattg gttnnnnnnn nn#nnnnnnnn    300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn    360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn    420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn    480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn    540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn    600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn    660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn#nnnnnnnn    720nnnnnnnnnn nnnnnnnnnn nnnnnnnntt cggatccttg gccaagtcaa ac#agtgtgaa    780ggaattacaa gtccagaagg ttccaaatcc atagtggaag gaatcataga gg#aggaggaa    840gaagatgagg aaggaagtga gtcagttaac aagaggaaaa aggaagacga ca#tggaaacc    900aagaaggatc acccatacac ctggagaatt gagctggcaa aaacagaaaa gt#actgggat    960ggctggttcc ggggcttatc caatctcttt cttagctgtc ctattcctaa ac#tgctgctc   1020ttggcgggtg ttgacagatt ggataaagat ctgaccatag gccagatgca gg#ggaagttc   1080cagatgcagg tcttacccca gtgtggccat gcagtccatg aggatgcccc tg#acaaggta   1140gctgaagctg ttgccacttt cctgatccgg cacaggtttg cagagcccat cg#gaggattc   1200cagtgtgtgt ttactggctg ctagtgacct gctgtctact cctccctcta ca#ttgagctc   1260tgttgtaaat acatcgcacc agaggccact gtgacgccgc tgtctcctcc tc#tccatccc   1320gcccagccat gtgacaccgg ctcttgtaga gggcatcccc agatgtccaa ac#cctttcct   1380gtgtactgtt gaaagcattg ttcttcaggg cccttgtcca acagtggccc gt#gcagtctg   1440gggtccacag ctcttcctct ccttcctgtg ctccctgcct tgcctaggat ga#agcctcca   1500gcgctgctcc ctggccctgt tcctggcata tggcaatgta ccccaggctc ag#ggatctcc   1560cttccttgag gatgttcttg gcatggtcct gccctacctc atgggatggg ca#atgcacac   1620actggccctt atttttccct ttcaaataaa acaccagtca ggtaccttta tc#ccagtctt   1680aactgtccca aatctggaag gtccagagta agcaggattc agggagaggg ag#tggatagc   1740aagtatccca agaaaccaac ctgtaagtca ggtccagcca gtccaagcac at#ggcttccc   1800atctgggtga gcccactgtc ccactcccac atgtctgggc acctgccctg gg#ctgaggcc   1860aggctgctcc aagggccgca tgagccctaa tctgccacag agcaacccag gt#taaacaca   1920gcccatgcac aaagccacag gctaaatcct gtggaattgt ttttaatgac tg#aatttaac   1980cattttcata gttggttcct ggaggtgtgc caagtgcccg cttgcctctt ct#agacccac   2040agcttcttga tccacttgtg gtttccatgt cactaatgta gaaacatcat gg#actagcat   2100ccccagtctt tgccctcatc cagctgtcgc agcgcacact ggggcctccc cc#tgctgccc   2160agggggggrc ggggtgggca gcctcctgaa acccatcttt ctgtgactgt ct#taggtgac   2220gtgtagccct cttccgtttt ttcacccaac aacttcctct gtcctgctgc ac#ggtccaga   2280gtctgggacc gactttgttt ctttgttatt tatgatcttg tttaaagaaa at#aaatatct   2340cccaaccttt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa#aaaaaaaa   2400 aaaaaaaaa                 #                  #                   #       2409 <210> SEQ ID NO 9 <211> LENGTH: 1202<212> TYPE: DNA <213> ORGANISM: Saccharomyces cerevisiae<400> SEQUENCE: 9tgtctgacga tttgagaaga aaaattgctt tatcccagtt tgagagagcc aa#gaatgttc     60tagatgcgac attccaagaa gcatacgagg atgatgaaaa cgatggtgat gc#attaggtt    120ccctgccatc atttaatgga caatcaaata ggaacagaaa atatacgggc aa#aaccggta    180gtactactga tagaatttca agtaaggaaa agagtagttt acccacttgg ag#tgattttt    240ttgataataa ggagttggta agtcttcctg atagagatct ggacgtaaat ac#atactata    300cattacctac ttcattgtta tcaaatacca cttcaattcc catctttatt tt#ccaccatg    360gggcgggctc ctcaggttta tcatttgcaa acttggccaa ggaattaaat ac#taaactag    420aaggaagatg cggatgcttt gcatttgatg ctagggggca tgcagaaaca aa#gtttaaga    480aggctgatgc gcctatatgc tttgacaggg actcttttat caaagatttt gt#aagcctgc    540taaattattg gtttaagtct aaaataagcc aagagccact tcagaaggta tc#tgttatac    600taattggtca ttcccttggt ggaagtatat gtacttttgc gtaccctaaa tt#atcaacag    660aactacaaaa gaaaattctt ggtattacta tgttagatat tgtagaagag gc#tgccatta    720tggccttaaa taaagttgaa cattttttgc agaatacacc caatgtattt ga#atcaatta    780atgacgctgt cgattggcac gttcaacacg cgttatcgag attgaggtca ag#cgccgaaa    840ttgctatacc agctttattt gctccgctca agtcagggaa agttgtcagg at#aacaaacc    900ttaagacctt tagccctttc tgggacacat ggtttaccga tctgtcgcac tc#ctttgttg    960gcttacctgt tagtaaatta ttaatattgg cgggaaacga aaatctcgat aa#agaattaa   1020ttgtggggca aatgcaaggt aaatatcagt tggtagtttt ccaagattcc gg#gcatttca   1080ttcaagaaga ttcgcctata aaaacagcaa tcactttaat tgatttctgg aa#gcggaacg   1140attctaggaa tgtagtaatc aagactaatt ggggtcaaca caaaaccgtg ca#aaatacat   1200 aa                   #                  #                   #            1202 <210> SEQ ID NO 10<211> LENGTH: 1095 <212> TYPE: DNA<213> ORGANISM: Caenorhabditis elegans <400> SEQUENCE: 10atgtccgacg ataaattaga cactcttccg gatcttcaat cggaaacgtc ac#atgtcaca     60actcctcaca ggcaaaatga tcttcttcgt caagcggtca ctcatggaag gc#caccacca    120gttccgagca catcaacttc tggaaagaaa cgagaaatgt ctgaactacc gt#ggtcagat    180ttttttgatg aaaagaagga cgcaaacatt gatggagatg ttttcaatgt gt#acataaag    240ggaaatgaag gtccaatttt ctatttgctt cacggtggag gttattcagg cc#tcacatgg    300gcgtgttttg cgaaagaatt ggcaacttta atatcatgca gagttgttgc ac#ctgattta    360agaggacacg gcgacactaa atgttctgat gagcacgatc tttcgaaaga aa#cccaaata    420aacgatattg gagcaatctt caagaacatt ttcggcgaag acgattcacc ag#tatgcatt    480gttggacaca gtatgggtgg tgcattggcc attcatacat tgaatgcaaa ga#tgatttct    540tcaaaagtcg ctgcactcat tgtcattgat gttgtcgaag gttccgctat gg#aagcactt    600ggaggaatgg ttcatttttt acattcaagg ccttcttcat ttccttctat cg#aaaaagcc    660attcactggt gcctttcttc gggtacagcg aggaatccca cagctgcacg gg#tctcaatg    720ccgtctcaaa ttagagaagt atcggaacac gagtacactt ggcgaattga tt#taacaaca    780acagaacagt actggaaagg atggtttgaa ggattatcca aagaattttt gg#gatgttcc    840gttccgaaga tgcttgttct agcgggcgtt gatcggctgg acagggatct ca#caattggt    900caaatgcagg gaaagtttca gacttgtgtg ttaccaaaag ttggacattg tg#ttcaggaa    960gatagcccac aaaatcttgc agatgaagtc ggaagattcg cttgccgcca ta#gaattgcc   1020caaccgaaat tctcagccct tgcatcacca ccagatccag cgattctcga at#acagaaaa   1080 cgtcatcacc aataa               #                  #                   #  1095 <210> SEQ ID NO 11 <211> LENGTH: 9<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 11Ile Met Leu Ile Gly His Ser Met Gly   1               5<210> SEQ ID NO 12 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Caenorhabditis elegans <400> SEQUENCE: 12Val Cys Ile Val Gly His Ser Met Gly   1               5<210> SEQ ID NO 13 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 13Val Ile Leu Ile Gly His Ser Leu Gly   1               5<210> SEQ ID NO 14 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Salmonella typhimurium <400> SEQUENCE: 14Leu Ile Ala Ile Gly Ala Ser Thr Gly   1               5<210> SEQ ID NO 15 <211> LENGTH: 10 <212> TYPE: PRT<213> ORGANISM: Artificial sequence <220> FEATURE:<221> NAME/KEY: UNSURE <222> LOCATION: (1)<223> OTHER INFORMATION: X at position 1 is  #Leu, Ile or Val.<223> OTHER INFORMATION: Description of Artificial  #Sequence: Signature      motif for lipases. <221> NAME/KEY: UNSURE <222> LOCATION: (2)<223> OTHER INFORMATION: X at position 2 is  #not specified as a      particular amino acid. <221> NAME/KEY: UNSURE <222> LOCATION: (3)<223> OTHER INFORMATION: X at position 3 is  #Leu, Ile, Val, Phe or Tyr.<221> NAME/KEY: UNSURE <222> LOCATION: (4)<223> OTHER INFORMATION: X at position 4 is  #Leu, Ile, Val, Ser or Thr.<221> NAME/KEY: UNSURE <222> LOCATION: (6)<223> OTHER INFORMATION: X at position 6 is  #His, Tyr, Trp or Val.<221> NAME/KEY: UNSURE <222> LOCATION: (8)<223> OTHER INFORMATION: X at position 8 is  #not specified as a      particular amino acid. <221> NAME/KEY: UNSURE <222> LOCATION: (10)<223> OTHER INFORMATION: X at position 10 is #Gly, Ser, Thr, Ala or Cys. <400> SEQUENCE: 15Xaa Xaa Xaa Xaa Gly Xaa Ser Xaa Gly Xaa   1               5 #                 10 <210> SEQ ID NO 16 <211> LENGTH: 51 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 16tgactacgtg cgtgcagcct cccctcgatg tcggccctcg aaaagagcat g #             51 <210> SEQ ID NO 17 <211> LENGTH: 7 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Sequence      resulting from alternative splicing. <400> SEQUENCE: 17Leu Leu Ser Thr Tyr Cys Arg   1               5

We claim:
 1. An isolated and purified polypeptide comprising the aminoacid sequence as given in SEQ ID NO:5.
 2. An immunogenic compositioncomprising one or more antigenic components, said antigenic component iseither an antigenic peptide linked to a carrier molecule to form apeptide-carrier complex or a multiantigenic peptide molecule having aplurality of antigenic peptides of identical amino acid sequence,wherein the amino acid sequence of the antigenic peptide consists ofamino acids 288-303 of SEQ ID NO:5.